Microbial compositions and methods

ABSTRACT

The present invention comprises compositions and methods for enhancing biological processes, such as plant growth or bioremediation. For example, the present invention comprises compositions and methods for effectively remediating chemical and organic wastes and reducing the environmental risk from manure, septic, sewage, oil pollution, and other contaminants.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/979,419filed Jan. 12, 2012, now Pat. No. 9,175,258, issued Nov. 3, 2015, whichis a U.S. national stage patent application under 35 U.S.C. §371 ofInternational Patent Application No. PCT/IB2012/000833 filed Jan. 12,2012 which claims priority under PCT Article 8 to U.S. application Ser.No. 61/432,152, filed Jan. 12, 2011 each of which is incorporated hereinby reference in its entirety.

TECHNICAL FIELD

The present invention relates to compositions and methods comprisingmicroorganisms, for example, for bioremediation. More particularly, thepresent invention comprises compositions and methods comprisingmicroorganisms, such as bacterial mixtures, for various purposes. Forexample, effectively remediating soil and water sources and reduction ofchemical and organic wastes and reducing the environmental risk frommanure, septic, sewage, oil pollution, and other contaminants.Additionally, the compositions and methods of this invention can be usedto sustainably manage soil and water, and repair ecosystems affected bycontamination or pollution.

BACKGROUND OF THE INVENTION

Contaminants, such as hazardous, polluting, or toxic materials orwastes, are a health and safety problem for the United States and forcountries world-wide. Governmental agencies, commercial companies, themilitary, and consumers are searching for more cost-effectivetechnologies that can be used to remove these hazardous and pollutingmaterials. Moreover, environmental regulations are mandating a change tosustainable management of soil and water contamination.

Contaminants are continuously produced by activities of humans on theplanet. In addition, natural causes can release or create contaminantsin the environment. Oil and radioactive elements are naturally releasedinto the environment and natural disasters, such as floods, createpolluted areas in their wake. In addition to the on-going production ofwaste materials, there is a large amount of contamination from human andnatural activities in the past.

The costs of cleaning up the environment are staggering. These costs area drain on the economies of thriving countries and are an almostinsurmountable problem for poorer countries. Many of the currenttechnologies for removing hazardous or contaminating wastes from theenvironment require transportation over long distances and involvesophisticated machinery or personnel, all of which add to the costs,environmental contamination, and do not support local communities.

Current methods of waste treatment are generally not adequate forremediating the contaminated air, soil or water. Chemical treatment canbe used on some wastes, but there may be hazardous by-products, leachateor sludge produced by treatment. Wastes may be isolated or alteredthrough methods such as stabilization, solidification or encapsulation.But, in these approaches the waste is merely contained, not destroyed orconverted. Additionally, the problem of storage of the contained wasteis created.

What is needed are compositions and methods that can performbioremediation, such as at a contaminated or polluted site, or toremediate materials such as soil or water that have an excess ofunwanted biological material or other contaminating or pollutingcompounds, or to enhance the health and sustainability of the localnatural environment, that can be applied to a wide variety ofenvironments. Even more ideally, would be a bioremediation system thatwould not only remove or stabilize the contaminated wastes, but wouldalso be capable of converting some of the wastes into usable products.What is also needed are methods and compositions for enhancingbiological processes, such as enhancing plant growth.

SUMMARY

The present invention comprises compositions and methods comprisingmicroorganisms, such as bacteria, for example, for treatments ofenvironments, for enhanced plant growth, reduction of contaminants,bioremediation, reduction of unwanted plant or microbial species, andproduction of natural products such as food supplements, nutriceuticals,cosmeceuticals cosmetics and pharmaceuticals. Compositions and methodsof the present invention may comprise microorganism-containingcompositions provided to an environment and/or microorganism-containingcompositions attached, adhered to, or in close association withsurfaces, wherein the surfaces can be living organisms, such asrhyzomes, rootlets and/or roots, or other plant or animal surfaces, orinert surfaces such as glass beads, shells, plant material, plastics,metal, wood, ceramics, and woven or nonwoven materials. The presentinvention comprises compositions and methods useful in the control andreduction of contamination and pollution in lentic water, such ascontained bodies of water, including but not limited to lakes and ponds.The present invention comprises compositions and methods useful in thecontrol and reduction of contamination and pollution in lotic water,such as flowing bodies of water.

The present invention comprises compositions and methods useful in thebioremediation of sites contaminated by hydrocarbons including, but notlimited to, oil and organic solvents, wherein site may comprise solids,soils, liquids, bodies of water, or mixtures of solids and liquids.

The present invention comprises compositions and methods useful inameliorating and cleansing hydrocarbon-contaminated wildlife. Thepresent invention comprises compositions and methods useful in thebioremediation of soil. The present invention comprises compositions andmethods useful in the treatment or management of commercial, municipaland residential septic systems, and reduction of biosludge.

The present invention comprises compositions and methods useful in thetreatment of municipal and industrial wastewater, the reduction offailures by treatment plants for municipal and industrial wastewater,and management of municipal or industrial wastewater. The presentinvention comprises compositions and methods useful in the recycling ofwater used in industrial, mining, commercial, private or individualprocesses.

The present invention comprises compositions and methods useful in thetreatment of water contained by dams.

The present invention comprises compositions and methods useful incontrolling odors generated by a site, and accelerating the microbialprocesses in composting facilities and for treating leachate fromlandfills.

The present invention comprises compositions and methods useful in thedecontamination of soils contaminated by hydrocarbons, solvents,pathogenic microbial organisms, animal wastes or other organic wastes.The present invention comprises compositions and methods useful in thedecontamination of soil and water, particularly contaminated soil orwater resulting from natural disasters or from people living inunsanitary conditions without adequate waste control facilities.

The present invention comprises compositions and methods useful in thetreatment of animal waste generated at facilities where animals arebred, raised, live or slaughtered, such as farms, and industrial farmingcomplexes for example, dairy farms in the production of milk, chicken oregg production facilities, swine production facilities, pet boarding andsitting facilities, and other commercial or individual farms or animalcontainment sites.

The present invention comprises compositions and methods useful in thecreation and maintenance of healthy soil environments. The presentinvention comprising compositions and methods useful to condition soiland maintain a balanced and sustainable soil ecology which can supportthe growth of plants, optionally with minimal chemical input. Thepresent invention enhances plant growth, supports plant maturation andincreased yields, rapid seed germination and increased biomass.

The present invention comprises compositions and methods useful in theremediation of mold, in contained environments, such as rooms orbuildings, or in exterior locations such as on surfaces of buildings,and for example such compositions and methods may be used in an areathat has been affected by flooding or in areas of high humidity.

The present invention comprises compositions and methods useful inimproving the qualities of soil. The present invention comprisescompositions and methods that aid in plant growth and enhance seedgermination, root production, increased yield by plants, increasedfruiting, prolonging production by plants either in a season orthroughout the productive lifespan of the plants, and increased vigorand biomass of plants.

The present invention comprises compositions and methods useful in thecontrol of insects and insect borne disease in plants. The presentinvention comprises compositions and methods useful in bee keeping.

The present invention comprises compositions and methods useful in thetechniques of hydroponic or aeroponic agriculture or a controlled plantgrowing system combining hydroponic and aeroponic methods. The presentinvention comprises compositions and methods useful in aquaculture orpisciculture. The present invention comprises compositions and methodsuseful in the process of vertical farming.

The present invention comprises compositions and methods useful in theremediation of industrial by-products, such as by-products from theconstruction industry, for example, treated lumber, from nuclear wastesfrom any source including defense or hospital sources, and otherindustrial wastes that may be treated with the compositions of thepresent invention. The present invention comprises compositions andmethods useful in the raising or keeping of animals.

The present invention comprises compositions and methods useful in themining industry. The present invention comprises compositions andmethods useful in the healthcare industry, for example by providingnatural sources of products extracted or purified from plants ormicrobial compositions or a combination thereof, comprising foodsupplements, nutriceuticals, pharmaceuticals, cosmeceuticals andcosmetics. The present invention comprises compositions useful asadditives in products for human or animal consumption or use.

The present invention comprises compositions and methods useful indomestic settings such as a household. The present invention comprisescompositions and methods useful in a variety of nanotechnologies such asnanoparticle delivery of microorganism-containing compositions s ormicroorganisms described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a chart of representative enzyme activity tests for certainbacteria.

FIG. 2 is a graph showing a decrease in coliforms after treatment with acomposition of the present invention.

FIGS. 3A and B are graphs showing nitrogen transfer (A) and nitrateincrease (B) in a site after treatment with a composition of the presentinvention.

FIG. 4 is a graph showing mercury levels in a site after treatment witha composition of the present invention.

FIG. 5 is a graph showing toluene levels in a site after treatment witha composition of the present invention.

DETAILED DESCRIPTION

The present invention comprises compositions and methods comprisingmicrobial organisms, which may be used in methods including but notlimited to, bioremediation and development and maintenance of healthyecosystems. Compositions may comprise a mixture of microorganisms,comprising bacteria, fungi, algae, and/or other indigenous or exogenousmicroorganisms, all of which form a micro ecosystem with roles for itsmembers. It is currently believed, though not wishing to be bound by anyparticular theory, that compositions and methods of the presentinvention enhance the naturally occurring mutual symbiosis between thefeeding roots of the plants, soil fungi and soil bacteria. Compositionsand methods of the present invention may comprise a mixture of isolatedmicroorganisms, and may act as a vehicle and/or a delivery system forexogenous microorganisms and/or endogenous consortia of microorganisms.

Compositions and methods of the present invention may act in a paracrineand/or mutuality regenerative fashion to restore a polluted orcontaminated environment's microorganisms and other flora. For example,in a composition of the present invention, one or more microorganismsmay be capable of modifying or eliminating the polluting orcontaminating compounds or molecules, one or more microorganisms mayprovide nutrition and promote healthy ecological processes, and one ormore microorganisms may facilitate detoxification activities andregenerate the natural ecology. Microorganisms of the present inventionmay consume specific substances in the polluted environment, and producemetabolic by-products that act as nutrients for other microorganisms ofthe composition of the present invention as well as to themicroorganisms existing in that environment. Compositions of the presentinvention may provide microorganisms that produce biological agentsincluding but not limited to antibiotics or surfactants.

Compositions of the present invention comprise isolated microorganismsand compositions comprising isolated microorganisms. A composition ofthe present invention is a mixed culture of isolated microorganisms(referred to as IN-M1) that was deposited with the ATCC under theBudapest Treaty, on Jan. 12, 2011, under Account No. 200139, and givenAccession Number PTA-12383. This composition comprises isolatedmicroorganisms Rhodopseudomonas palustris, Bacillus subtilis,Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillis helveticusand Lactobacillus caseii. Compositions of the present invention comprisecompositions comprising one or more isolated microorganisms. Forexample, a composition may comprise Rhodopseudomonas palustris, Bacillussubtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillusoryzae, Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus, and Lactobacillus caseii. For example, acomposition may comprise the isolated microorganism Lactobacillishelveticus, referred to herein as IN-LH1, which was deposited with theATCC under the Budapest Treaty, on Jan. 12, 2011, under Account No.200139, and given Accession Number. PTA-12386. A composition maycomprise the isolated microorganism Bacillus subtilis, referred toherein as IN-BS1, which was deposited with the ATCC under the BudapestTreaty, on Jan. 12, 2011, under Account No. 200139, and given AccessionNumber. PTA12385. A composition may comprise the isolated microorganismSaccharomyces cerevisiae, referred to herein as IN-SC1, which wasdeposited with the ATCC under the Budapest Treaty, on Jan. 12, 2011,under Account No. 200139, and given Accession Number. PTA-12384. Acomposition may comprise the isolated microorganism Rhodopseudomonaspalustris, referred to herein as IN-RP1, which was deposited with theATCC under the Budapest Treaty, on Jan. 12, 2011, under Account No.200139, and given Accession Number. PTA-12387. A composition of thepresent invention may comprise a mixture of isolated microorganismscomprising one or more of Lactobacillis helveticus, referred to hereinas IN-LH1 (Accession No. PTA-12386), Bacillus subtilis, referred toherein as IN-BS1(Accession No. PTA-12385), Saccharomyces cerevisiae,referred to herein as IN-SC1 (Accession No. PTA-12384), Rhodopseudomonaspalustris, referred to herein as IN-RP1 (Accession No. PTA-12387).Examples of isolated microorganisms in compositions of the presentinvention include, but are not limited to, Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and/or Lactobacillus caseii.Compositions of the present invention may comprise differing amounts andcombinations of these and other isolated microorganisms depending on themethods being performed. Compositions of the present invention areuseful in the methods taught herein.

Isolated microorganisms that are useful in compositions and methods ofthe present invention include, but are not limited to, one or more ofthe following:

Microorganisms Lactobacillus Hyperthermophile Lactobacillus fermentumMethanopyrus. kandleri Streptococcus thermophilusMethanobrevibactersmithii Lactococcusdiacetyllactis PyrococcusfuriosusLactococcuslactis Ferrglobus Bifidobacteriumbifidum FerrglobusplacidusLactibacillusdelbruecki Hydrothermal Yeasts Pyrolobusfumarii Candidaantarctica Thermophile Candida chauliode SulfolobusacidocaldariusCandida corydali Sulfolobusislandicus Candida albicansSulfolobusmetallicus Lodderomyceselongisporus. SulfolobusshibataeCandida dosseyi Sulfolobussolfataricus Candida blattae Bacillusthuringiensis Candida ascalaphidarum Bacillus Candida membranifaciensthuringiensisIsraelensis Candida oleophila Pseudomonas Streptomycesalbus Pseudomonas alcaligenes Lachanceafermentati, Pseudomonas mendocinaLachanceathermotolerans Pseudomonas pseudoalcaligenesHanseniasporavineae Pseudomonas resinovorans SaccharomycotinaPseudomonas veronii Aspergillus Pseudomonas putida AspergillusoryzaePseudomonas stutzeri Aspergillusniger Pseudomonas fluorescensAspergillusterreus Pseudomonas chlororaphis AspergillusfischerianusPseudomonas aurantiaca Green sulfur bacteria Pseudomonas aeruginosa,Purple sulfur bacteria White rot fungi Chromatiaceae XanthomonasEctothiorhodospiraceae, Acinetobacter Halothiobacillaceae Rhodococcussp. Halothiobacillushalophilus ArthrobacterHalothiobacillushydrothermalis Aureobasidium sp. HalothiobacilluskellyiAlcaligeness sp. Halothiobacillusneapolitanus Leuconostoc sp. Purple nonsulfur bacteria Sclerotium sp. Rhodopseudomonas palustris Clostridium,Salt or Ocean Bacterium Zymomonas Halobacteriumjilantaiense KlebsiellaHalobacteriumnoricense Micorrhizal fungi HalobacteriumsalinarumHalobacteriumpiscisalsi

The present invention comprises isolated microorganisms. For example, anisolated microorganism of the present invention is Lactobacillishelveticus, referred to herein as IN-LH1, which was deposited with theATCC under the Budapest Treaty, under Account No. 200139, and givenAccession Number. PTA-12386. An isolated microorganism of the presentinvention is Bacillus subtilis, referred to herein as IN-BS1, which wasdeposited with the ATCC under the Budapest Treaty, under Account No.200139, and given Accession Number. PTA-12385. An isolated microorganismof the present invention is Saccharomyces cerevisiae, referred to hereinas IN-SC1, which was deposited with the ATCC under the Budapest Treaty,under Account No. 200139, and given Accession Number. No. PTA-12384. Anisolated microorganism Rhodopseudomonas palustris, referred to asIN-RP1, which was deposited with the ATCC under the Budapest Treaty,under Account No. 200139, and given Accession Number. PTA-12387.

In an aspect, the isolated microorganisms of the present invention canbe grown in large, industrial scale quantities. For example, and not tobe limiting, a method for growing microorganisms in 1000 liter batchescomprises media comprising 30-70 liters of non-sulphur agriculturalmolasses, 2-5 liters of wheat bran (0.02-0.05% by volume), 2-5 liters ofkelp (0.02-0.05% by volume), 2-5 liters of bentonite clay (0.02-0.05% byvolume), 0.5 to 3(0.005-0.03% by volume) liters fish emulsion (acommercially available organic soil amendment, from Nutrivert, Dunham,Quebec non-pasteurized), 0.5 to 3 liters soy flour(0.005-0.03% byvolume), 0.4 to 1.5 mg of commercially available sea salt, and 20-70liters total of selected strains of isolated microorganisms at 1×10⁵ to1×10⁷ cells/mL, for example, Lactobacillis helveticus, (IN-LH1,Accession Number. PTA-12386), Bacillus subtilis, (IN-BS1, AccessionNumber PTA-12385), Saccharomyces cerevisiae, (IN-SC1, Accession Number.PTA-12384), Rhodopseudomonas palustris, (IN-RP1 Accession Number.PTA-12387), or a mixed culture, IN-M1 Accession Number. PTA-12383.

The total is brought to 1000 liters in non-chlorinated warm water. Amethod for growing the microorganisms may comprise dissolving molassesin some of the warm water, adding the other ingredients to the filltank, adding the desired bacteria, keeping the temperature between28-35° C., and, after the pH drops to about 3.5-4.0, usually withinseveral days, with stirring lightly and monitoring of pH. The culturecan incubate for weeks, typically from 5 to 10 weeks, and may bebottled, or stored, resulting in a composition of the present invention.The composition may be bottled and stored, for example in airtightcontainers out of sunlight at room temperature. In one aspect, theculturing method and composition may include samples of a pollutant orpollutants from an environment to be remediated or restored, andoptionally, the sample may include indigenous microorganisms from thepolluted environment. Compositions may comprise one or a consortium ofendogenous microorganisms isolated from the environment to be treatedand/or remediated.

The term, “remediation” as used herein, is the act or process ofcorrecting a fault or deficiency, such as in modulating, ameliorating,reducing, reversing or stopping environmental damage. In the case ofenvironmental remediation, it is acting on contaminants, such ashazardous or polluting materials, which may be changed chemically, orphysically, or stabilized or sequestered or in some other way removedfrom the surrounding environment. Remediation may comprise reduction ofphosphates and may include the combination compositions of the presentinvention with plants, endogenous and exogenous natural microorganismsand animalia.

Bioremediation, as used herein, means using biological organisms, aloneor in conjunction with inert structures, as a system for treating,modulating or altering the contaminants, such as hazardous or pollutingmaterials.

Contaminant, as used herein, means any molecules, chemicals or organismsin the environment that are harmful to other living organisms in theenvironment or to the abiotic elements of the environment, and includescompounds or molecules that are in an amount greater than is desired forthat environment even if such compounds or molecules are not inherentlyharmful if found in lower amounts, and the term may be usedinterchangeably with the term “pollutant”. Biological, chemical,physical, or radiological substance (normally absent in the environment)which, in sufficient concentration, can adversely affect livingorganisms through air, water, soil, and/or food are included in theterms contaminant or pollutant. The term, “toxic materials” as usedherein, is included in the term contaminant. The contaminants may alsobe a natural element of the environment that is present in such aconcentration that it is now harmful to the environment and itsconstituents. The contaminant may be an element that has been introducedinto the environment by human activities, such as synthesis of thematerial, or by natural causes. The term contaminant, as used herein,encompasses the presence of one or more toxic, hazardous, or pollutingmaterials in an area.

Contaminant, as used herein, also means any molecules, chemicals ororganisms in the environment that are present in an undesiredconcentration or amount. The contaminant may not necessarily be harmingany component of the environment but may be present in an undesiredquantity.

The term, “environment” as used herein, is defined generally as thesite, surroundings or conditions in which a person, animal, or plantlives or operates, and more specifically in terms of remediation as anarea as defined by the contaminant situation, and environment mayinclude the biotic and abiotic elements, and the patterns of theinterrelationships between the biotic elements, and between the bioticand abiotic elements which are found in the defined area. All threephysical states, solids, liquids and gases, may be included in theelements that make up the environment.

As used herein, microorganism include, but are not limited to, bacteria,viruses, fungi, algae, yeasts, protozoa, worms, spirochetes,single-celled and multi-celled organisms that are included inclassification schema as prokaryotes, eucaryotes, Archea, Bacteria andthose that are known to those skilled in the art. Microorganisms mayalso refer to isolated microorganisms and may comprise particulardeposited compositions or microorganisms disclosed herein, and theintent of the text can be interpreted by those of skill in the art.

The compositions of the present invention comprise a combination ofisolated microorganisms from several genera and/or species. Theseisolated microorganisms grow and live in a cooperative fashion, in thatsome genera or species may provide by products or synthesized compoundsthat are beneficial to other microorganisms in the combination. Inmaking a mixed culture, individual characteristics of one or moreisolated microorganisms are used to select a microorganism for inclusionin the mixed culture. For example, characteristics may include, but arenot limited to, enzymes made by the microorganism, the ability tosupplement another microorganism's metabolism, growth or otheractivities; the ability to complement the metabolic or enzymaticpathways of other microorganisms in the mixed culture, non-sporeformation, ability to survive, replicate or metabolize in adverse mediaor conditions, oxygen consumption, ability to use energy sources such aslight or chemicals other than carbon, the lack of predation on othermicroorganisms in the mixed culture, the ability to provideenvironmental aspects, such as structures (by, for example, producinghyphae, cellular extensions, pilli, or forming a slime or biofilm layeron structures in the environment), pH, reducing or enhancing oxygenlevels, carbon dioxide levels, or other metabolic needs, and otheractivities known to be made by one microorganism that can be used bymicroorganisms in the mixture. Those of skill in the art in themicrobial arts are well aware of such characteristics of microorganisms.One or more mixed cultures may be used in compositions and methods ofthe present invention, for example, IN-M1, or compositions comprisingIN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination or individually,and/or with other isolated microorganisms, such as those isolated from asite to be treated or sites similar to those to be treated.

For example, in a composition of the present invention, both aerobicmicroorganisms, which need oxygen for metabolic activities, andanaerobic microorganisms, which use other sources of energy such assunlight or the presence of specific substrates, are combined to form acomposition. This enables the composition's isolated microorganisms tocolonize substrates in different regions of an environment. For example,one or more mixed cultures may be used in compositions and methods ofthe present invention, for example, IN-M1, or compositions comprisingIN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination or individually,and/or with other microorganisms.

A composition may comprise facultative microorganisms, for example,strains of lactobacillus, which modulate metabolic activities accordingto oxygen and/or nutrient concentrations in the environment. Though notwishing to be bound by any particular theory, it is currently believedfacultative microorganisms in a composition provide a prolongedshelf-life at room temperature for a composition of the presentinvention. For example facultative microorganisms such as IN-BS1 orIN-LH1 may be included in compositions and methods described herein.

All species of living organisms include individual organisms that varygenetically and biochemically from each other but are still within whatis called the spectrum of normal variations within the species. Theseindividual natural variations maybe the result of nondisruptivesubstitution or deletions in the gene sequence, variation in geneexpression or RNA processing and/or variations in peptide synthesisand/or variation of cellular processing of intra cellular, membrane orsecreted molecules. Compositions of the present invention may comprisemicroorganisms that are within or without the normal variations of aspecies. Identification of such microorganisms may be detected bygenetic, molecular biological methods known to those skilled in the art,and/or by methods of biochemical testing.

For example, a composition of the present invention comprises isolatedmicroorganisms selected by isolating individual colonies of a particularmicroorganism. The colony members were characterized, for example, byone or more characteristics, such as by testing enzyme levels present inthe isolated microorganism and the activity with particular substratesin a panel of substrates, to establish an enzyme profile for theisolated microorganism. These substrates are representative of across-section of the biochemical pathways needed by the microorganismsin a composition of the present invention that is to be used in forgeneral remediation purposes, such as the breakdown of organic matter ofvegetable or animal matter, or for specific remediation purposes, suchas the breakdown of particular chemicals. One or more selection criteria(characteristics of a microorganism) enable the formulation ofcompositions of isolated microorganisms that provide a standardizedecosystem that is ready to colonize an environment and to carry outparticular reactions. For example, one characteristics is enzymes andactivity of enzymes in microorganisms, such that all of themicroorganisms that provide enzymes were tested for enzyme activitylevels for substrates, enzyme profile testing. Microorganisms havingcompatible enzyme profiles may be combined to provide a composition formethods disclosed herein. Complementary enzyme profiles means that theenzyme profile of one microorganism is different from the enzyme profileof another microorganism, for example, one microorganism has fataseactivity and another does not, or one microorganism providing an enzymein a metabolic pathway that is lacking in another organism, or onemicroorganism have cellulase activity of +5 and another microorganismhaving a cellulase activity of +3 or +4.

An example of an enzyme profile test comprises providing substrates andnoting where there is high activity, +3 or greater, and where there islittle to no activity, +2 and below. For example, a lactobacillus wastested and had a +5 enzyme level for alkaline phosphatase and a 0 forlipase. Such a lactoballicus may be admixed in a composition of thepresent invention with another microorganism where the lipase productionis+4 or +5. For example, a lactobacillus such as IN-LH1 may be used.Both enzyme activities may be desired in compositions where reactionsare needed to breakdown organic matter, for example. Usingmicroorganisms with differing characteristics allows for cooperative,completion of metabolic pathways in order not to have incompletebiochemical pathways which leave, either intermediary compounds that arenot bioavailable to the environment, or are not available to othermicrobial species as nutrition or prebiotics, or that do not provideactive compounds/enzymes targeting specific pollutants. Incompletebiochemical pathways also do not provide molecules involved in theproduction or activation of nutritional elements for different speciesin the composition or in the inoculated environment; hormones, growthfactors, anti-bactericides etc. with paracrine influence on the growthand regeneration of the damaged environment. Another detrimental effectof incomplete biochemical pathways are the production of intermediarycompounds that are ether toxic and/or odorous, like hydrogen sulfide.

An example of an enzyme profile test for isolated microorganisms of thepresent invention is shown in FIG. 1.

For example, bacterial strains isolated from Bacillus species werecharacterized by enzyme profiles comprising ability to hydrolyze fibrousmatter (cellulose and hemi cellulose), proteins and fat; and ability tocomplete the degradation of numerous intermediary molecules and cellsthat accumulate during the metabolic processes. These characteristicsare useful for degradation of organic matter and reduction or preventionof odors and other gas emissions. On a scale of 0 to 5, by trained eyesin assessing the color change in the enzyme reaction, the ranking +4 to+5 are generally optimal, though lower responses by particular organismsmay be acceptable. For example, a deposited culture of Bacillus subtilisIN-BS1, may be used as described herein.

The enzyme tests are commercially available and the testing proceduresfor microorganisms and methods for determining activity levels are wellknown in the art. Other characteristics include, for example, screeninga yeast, such as Saccharomyces cerevisiae for fermentation enzymes andBacillus species with known enzyme profiles. For example, a depositedculture of Saccharomyces cerevisiae microorganism, IN-SC1, may be usedas described herein. Testing microorganisms to determine which strainshave high enzyme activity of certain enzymes which take part in provenfermentation pathways allows for identification of microorganisms thatwill perform well in compositions used in methods for digesting organiccomponents in food and which can use the available nutrients of organicorigin; Isolation of individual colonies and the enzyme profile testingof these allows for the isolation of strong expressers of the enzymes.Additionally, using a combination of microorganisms that providecomplimentary characteristics allows for complete degradation of organicmatter, even in a cross-species manner. Though not wishing to be boundby any particular theory, it is believed that in the environment, as inhigher organisms including humans, both one-way and two-way interactionsbetween different cells may contribute to complex networks ofinterdependencies in microbial ecosystems.

Also not wishing to be bound by any particular theory, it is believedthat identifying and using microorganisms that provide complementary orcompatible profiles, such as enzyme profiles or other activities, allowsfor the inclusion of species that are indigenous to a site that istreated by the present invention. Other functional screening tests canbe used to characterize indigenous complementary or compatiblecharacteristics criteria for compositions that are used in remediationof particular pollutants and for the identification of other indigenousmicroorganisms.

An aspect of microorganisms which may be included in compositions of thepresent invention may be the characteristic of the formation or lack offormation of spores (non-sporulation). For example, bacterial isolatesmay be selected based on their response when moved from a starvationmedia to a nutrient rich media. isolates that show aggressive growthwhen transferred from a starvation medium to nutrient rich medium, andthat also showed decreased sporulation, or a lower amount of sporeformation in the starved cultures, are sometimes optimal forcompositions. A species that survive adverse conditions by formingspores may or may not be optimal for compositions of the presentinvention. For example, species that sporulate less and rest in avegetative state in adverse environments and then show aggressive growthin numbers in optimal environments may be more beneficial incompositions for remediating organic wastes and other contaminants.

An aspect of a characteristic for selection of microorganisms which maybe included in a composition of the present invention is growth of themicroorganism at different temperatures. For example, a Bacillus strainmay be selected based on its ability to grow at different temperaturesin aerobic conditions. Growth curves numbers of bacteria by OD and pHover time at different temperatures from 15 C-40 C may be used as anaspect for selection. Selected strains may be capable of growth in thepresence of nitrates, capable of growth under anaerobic conditions orhave other selected characteristics. For example, a Bacillus or purplenon sulfur bacteria useful in methods and compositions of the presentinvention is characterized by the following: a +5 level of cellulase, a+2 to +3 level of proteinase, at least a +4 of fatase, grows in an 8%nitrate media, grows in a range of temperatures from 30° C. to 40° C.,and does not form spores. For example, deposited cultures IN-BS1and/orIN-RP1 that comprise bacillus and purple non sulfur bacteriamicroorganisms, respectively, are used as described herein.

B. subtilis will grow anaerobically, either by using nitrate or nitriteas a terminal electron acceptor, or by fermentation. A two-componentsignal transduction system is an early stage in the regulatory pathwaygoverning anaerobic respiration. One of the essential roles of ResD andResE in anaerobic gene regulation is induction of fnr transcription uponoxygen limitation. FNR is a transcriptional activator for anaerobicallyinduced genes, including those for respiratory nitrate reductase,narGHJI. B. subtilis has two distinct nitrate reductases, one for theassimilation of nitrate nitrogen and the other for nitrate respiration.In contrast, one nitrite reductase functions both in nitrite nitrogenassimilation and nitrite respiration. Unlike many anaerobes, which usepyruvate formatelyase, B. subtilis can carry out fermentation in theabsence of external electron acceptors wherein pyruvate dehydrogenase isutilized to metabolize pyruvate. B. subtilis generally grows at 25-37°C., gene expression observed at 15° C.-40° C. For example, depositedcultures that comprise a B. subtilis, IN-BS1 with ATCC number PTA-12385are used as described herein.

Methods for selection of a microorganism for compositions describedherein comprise determining characteristics of microbial isolates andestablishing a profile of the characteristics for each isolate, such asby testing for enzyme profile activity, growth characteristics underdiffering conditions such as oxygen levels or temperature, growth inparticular media conditions, such as nitrates, carbohydrates, mineralsor particular contaminants, and characteristic responses for particularmicroorganisms, such as the ability to form spores or the ability togrow in the presence of pollutants or contaminants. Such tests ofmicrobial characteristics are known in the art and any known tests arecontemplated by the present invention. These tests allow for thecharacterization of a particular microorganism, the establishment of aprofile of the characteristics of a particular microorganism, and amethod of making a useful composition of the present invention comprisesselecting microorganisms that are compatible individual species, basedon their individual characteristics and how they interact together in amixture. For example, two microbes are complementary when each one'scharacteristic profile is different in at least one characteristic fromthat of the other microbe, or complementary may comprise wherein acharacteristic of one microbe is beneficial to or interactive with acharacteristic of a second microbes or other microbes in the mixture. Asused herein, compatible may mean that microorganisms can generallycoexist in a composition. One testing criteria (characteristic) formicroorganisms of the present invention in making a mixed culture is theinteraction of the various species together. A microorganism may havethe enzyme profile, or other tests and other characteristic criteria tomake it compatible with at least one other microbe, but the firstmicroorganism may not be able to grow well when added to a consortium ofmicroorganisms, and thus that microorganism would not be selected as acomponent of a mixed culture composition of the present invention.Characteristics disclosed herein may be used to select individualmicroorganisms, or may be used to characterize a mixed culture ofmicroorganisms as to the suitability of the mixed culture.

As used herein a microorganism does not mean one individualmicroorganism but a population or plurality of identical microorganism,such as a colony of B. subtilis. A mixture of microorganisms is amixture of different microorganisms, such as B. subtilis, L. helviticus,L. casei, and a mixture may comprise different species, strains, genera,types, or other recognized ways of classifying, separating oridentifying microorganisms.

Selection criteria of oxygen metabolism and nutrient concentrations maybe used to characterize microorganisms in a composition of the presentinvention. For example, Lactobacillus strains may be selected based onthe ability to modulate metabolic activity depending on the oxygenconcentration or nutrient concentration in the growth media, and byextension, what activities the lactobacilli will have in a particularenvironment when used in a composition of the present invention. Forexample, Lactobacillus convert lactose and other sugars to lactic acid.The production of lactic acid makes the lactobacillus environmentacidic, which inhibits the growth of some harmful bacteria. The majorityof acidifying flora in the culture generally control the pH of theenvironment. This characteristic allows for methods of the presentinvention, when remediating an environment, to provide an environmentwith low pH without having to provide a very high volume of acomposition. Such compositions can provide high concentrations of theacidifying microorganisms and provide a composition that has a greaterthan two years shelf life. In certain Lactobacillus strains, forexample, L. plantarum, the secretion of lactic acid is down regulatedwhen the pH is below a pH 3 and up regulated when the pH is too high,whereas pH 4-5 is optimal.

Lactobacillus strains for a composition of the present invention may beselected on the basis of their ability to help provide the components inthe formulation that provide stability for a prolonged shelf-life atroom temperature. For example, if a composition of the present inventionis stored in a sealed or closed container at room temperature, thebiological activity of that composition is measured by opening thecontainer and reactivation of the composition by known microorganismsteps of dilution and the addition of molasses (a nutrient source) andincubation at 30-37° C. This is monitored by observing the pH going downto 3.7 and below in 5-7 days. A sealed container of a composition of thepresent invention, stored at room temperature, has a shelf life oflonger than 2 months, longer than 4 months, longer than 6 months, longerthan 8 months, longer than 10 months, longer than 1 year, or one year orlonger. For example, deposited cultures that comprise Lactobacillusspecies, IN-LH1 with ATCC number PTA-12386 are used as described herein.

Beneficial yeasts, fungi and aspergillum provide nutrition, secretedenzymes and a network which provides filamentous structures in soil andwhich provide a structure to water or other liquids for biologicalcomponents of a composition and the endogenous bacteria. Beneficialyeasts are those yeasts that are not pathogenic to plants or animals.Though not wishing to be bound by any particular theory, it is believethat providing structure, as mycelium does, increases the ability of thevarious species to find their preferred environment in the remediationenvironment and maximize their colonization and bioactivity. In acomposition of the present invention, yeasts, fungi and aspergillumprovide filamentous structures for the components to populate and celldebris to maintain the anaerobic photosynthetic bacteria present in theformulation.

A characteristic for yeast or micorrhyzi fungus that may be important inparticular uses of compositions of the present invention is that theyare nonpathogenic and nontoxic to humans and animals. It is known thatmycelium in soil is important for aeration and that it excretesexoenzymes that breakdown organic nutritional sources for the fungi andthat are used by the microbes in the environment. Fungi bind heavymetals such as cesium and other elements and are known to be useful inthe purification of water flowing through the mycelium. It is believedthat the mycelium attract symbionts (Bacillus sp., Pseudomonas sp. etc)in the environment and provide a fibrous network to colonize in acoexistence and providing an exocellular cooperative source ofnutrients. For example, compositions that comprise yeast such as IN-SC1or species expressing an enzyme system that includes cytochrome P450protein or domain and/or fusaric acid or soil micorrhizal fungi are usedas described herein.

The present invention comprises compositions comprising photosyntheticbacteria. For example, R. palastrus, such as IN-RP1 (AccessionNo.PTA-12387) may be a component of a composition based on its abilityto scavenge organic waste, toxins and hydrocarbons.

Phototropic bacteria may be components of compositions of the presentinvention. Purple sulfur or nonsulfur bacteria may be used in thecompositions of the present invention. For example, compositions thatcomprise photosynthetic bacteria, IN-RP1 with ATCC number PTA-12387 areused as described herein.

Compositions of the present invention comprise bacteria and othermicroorganisms, such as those shown in Table 1. Bacteria which may beuseful in the present invention include, but are not limited to thefollowing. Bacillus alcalophilus, Bacillus alvei, Bacillusamyloliquefaciens, Bacillus aneurinolyticus, Bacillus aquaemaris,Bacillus brevis, Bacillus caldolyticus, Bacillus centrosporus, Bacilluscereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus flavothermus, Bacillus fusiformis, Bacillusglobigii, Bacillus halodurans, Bacillus infernos, Bacillus larvae,Bacillus laterosporus, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus mesentericus, Bacillus mucilaginosus, Bacillusmycoides, Bacillus natto, Bacillus pantothenticus, Bacillus polymyxa,Bacillus pseudoanthracis, Bacillus pumilus, Bacillus schlegelii,Bacillus sphaericus, Bacillus sporothermodurans, Bacillusstearothermophilus, Bacillus subtilis, Bacillus thermoglucosidasius,Bacillus thuringiensis, Bacillus vulgatis, and Bacillusweihenstephanensis.

The present invention comprises compositions and methods useful in thecontrol and reduction of contamination and pollution in lakes and ponds.More specifically, the present invention comprises compositions andmethods useful in the control and reduction of pond sludge. As usedherein, “pond sludge” can mean algae, duckweed, or any other organicmatter on the surface of the pond or underneath the surface of the pond.The compositions may comprise a blend of beneficial bacteria, yeasts andfermentation products, selected to grow with a particular species ofmicroorganism, for example, bacillus species, and to enhance the growthor activities of natural microbes present in biological and organicwaste and the surrounding environment. Compositions for control andreduction of contamination or pollution in lakes and ponds may compriseone or more mixed cultures used in compositions and methods of thepresent invention, for example, IN-M1, and/or compositions comprisingIN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination or individually,and/or with other microorganisms.

The compositions may be a concentrated solution of microorganisms, forexample, 1×10⁹ cells per L, and can either be used as a concentrate, ordiluted, for example at a rate of from about 1×10¹ to about 1×10⁴, andsprayed on the surface and edges of the body of water or simplyactivated (placed in nutrient media and allowed to grow from an aliquotof a concentrated solution diluted in media) in an activation center(container). An activation center is one or more containers for growinga composition. A sealed or closed container of a composition is providedto a treatment location and the container is opened, the composition maybe diluted into a media in an activation container or containers and themicroorganisms are grown in media at 30° -37° C. for several days, suchas 5-7 days, or until the pH is measured at pH 4, or 3.7, or below. Thisactivation of the composition does not need to be performed understerile or clean conditions, and it is beneficial if it is done on sitewhere the remediation will take place. This is beneficial for watertreatment methods. An activation center can be used, for example, forbatches of 200-1000 liters and can be a modular design that can easilybe expanded to increase activation capacity. The modules may be 200-1000liters in size and multiple modules may be used to activate acomposition. Activation of a composition is not limited to use of anactivation container, and the composition may be grown in any containerof a size needed to adequately allow the microorganisms in thecomposition to reproduce and survive.

In one aspect, the method of activation comprises 1) closing the valveon the activation container, 2) putting 10 liters of media into thecontainer, 3) adding 20 liters of water, 4) adding 10 liters of themicroorganism composition, 5) filling the activation container withadditional water to the 200 liter mark, 6) heating to 30° -37° C.)leaving for 7-10 days, 8) testing the pH, and 9) using the activatedcomposition, for example for application to a site once the pH is at orbelow 3.7. In one aspect, the bodies of water can be interconnectingshallow ponds. In one aspect, the compositions and methods are useful inthe control of odor resulting from the accumulation of sludge at thebottom of a body of water. Methods of treating lentic water, such asponds, lakes or contained bodies of water comprise methods of using amicroorganism-containing composition comprising applying an effectiveamount of a microorganism-containing composition to lentic water or acontained body of water, allowing the composition to remain for a timeperiod, for example time to grow and reproduce at the treated site, andreducing or eliminating at least a portion of the polluting orcontaminating materials.

The present invention comprises compositions and methods useful in thecontrol and reduction of contamination and pollution in flowing bodiesof water such as rivers, streams, underground aquifers or undergroundstreams. Compositions may comprise a microorganism-containingcomposition comprising Rhodopseudomonas palustris, Bacillus subtilis,Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae,Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus and Lactobacillus caseii.Microorganism-containing compositions may comprise one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a concentrated composition as taught inExample 1 may be used. For example, a microorganism-containingcomposition may comprise IN-M1, Accession No. PTA-12383. Methods ofusing a microorganism-containing composition comprise applying aneffective amount of a microorganism-containing composition to flowingwater, allowing the composition to remain for a time period, for exampletime to grow and reproduce at the treated site or in the flowing water,and reducing or eliminating at least a portion of the polluting orcontaminating materials. Methods of using a microorganism-containingcomposition to treat flowing water may comprise providing a structurecomprising an attached or a contained microorganism-containingcomposition such that the flowing fluid contact the immobilizedmicroorganism-containing composition, having the microorganisms act onthe flowing fluid, and reducing or eliminating one or more polluting orcontaminating materials in the flowing fluid or lotic water.

The present invention comprises compositions and methods useful in theremediation of sites contaminated by hydrocarbons and/or heavy metalsand/or unwanted bacteria such as E. coli. In one aspect, thecompositions can be used to convert toxic hydrocarbons into harmlesscompounds of nitrogen, carbon dioxide, and water. Compositions maycomprise a microorganism-containing composition wherein themicroorganisms comprise Rhodopseudomonas palustris, Bacillus subtilis,Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae,Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus and Lactobacillus caseii. For example, aconcentrated composition as taught in Example 1 may be used.Microorganism-containing compositions may comprise one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a microorganism-containing composition maycomprise IN-M1, Accession No. PTA-12383. Methods of using amicroorganism-containing composition comprise applying an effectiveamount of a microorganism-containing composition to a site or containercomprising hydrocarbons and/or heavy metals and/or E. coli. allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and reducing or eliminating atleast a portion of the hydrocarbons and/or heavy metals and/ormicroorganisms such as E. coli.

The present invention comprises compositions and methods useful in themining industry, In one aspect the compositions and methods can be usedto clean or treat mine tailings. In one aspect the compositions andmethods can be used to clean or treat wastewater generated by mining.Compositions maybe delivered using existing equipment, in fresh water,salt water or briny conditions, aerobic and anaerobic conditions andinterfacing with existing infrastructure, existing and new aeration,mixing separation and monitoring systems used in the treatment ormanagement of contaminated water, sludge, soil and mixtures thereof.Compositions may comprise a microorganism-containing composition whereinthe microorganisms comprise Rhodopseudomonas palustris, Bacillussubtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillusoryzae, Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus and Lactobacillus caseii. For example, aconcentrated composition as taught in Example 1 may be used.Microorganism-containing compositions may comprise one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a microorganism-containing composition maycomprise IN-M1, Accession No. PTA-12383. Methods of using amicroorganism-containing composition comprise applying an effectiveamount of a microorganism-containing composition to a site or containercomprising contaminants resulting from mining activities, allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and reducing or eliminating atleast a portion of the contaminants resulting from mining activities,such as mine tailings, waste waters, soils or equipment.

The present invention comprises compositions and methods useful in thebioremediation of sites, containers or water sources contaminated byhydrocarbons, such as oil. In one aspect the contamination can resultfrom an oil spill. Compositions of the present invention may comprisemicroorganisms that process hydrocarbons and nutrients which canstimulate the growth of microorganisms in the polluted environment toaid in reestablishment plant life in the affected environment and toprotect marine and other wildlife that may be affected by the oil spill.Compositions comprising microorganisms may degrade hydrocarbons andother pollutants found at oil spill sites, like organic solvents, andmay do so even in the presence of chemicals like surfactants. A methodof the present invention may comprise applying or providingmicroorganism-containing compositions comprising oil degrading microbialorganisms to a site contaminated by oil or other hydrocarbons, allowingthe composition to remain for a time period, for example time to growand reproduce at the treated site or surface, and removing at least aportion of the oil or hydrocarbons present. Compositions may comprisemicroorganisms adapted to degrade hydrocarbons, for example, isolatedmicroorganisms that were isolated from an oil or hydrocarboncontaminated site, in combination with microorganism-containingcompositions taught herein. In one aspect, a composition can be sprayeddirectly onto the oil at the site of contamination. Compositions maycomprise a microorganism-containing composition comprisingRhodopseudomonas palustris, Bacillus subtilis, Pseudomonas aeruginosa,Saccharomyces cerevisiae, Aspergillus oryzae, Candida utilis,Streptococcus lactis, Lactobacillus caseiiLactobacillis planterum,Lactobacillis helveticus and Lactobacillus caseii.Microorganism-containing compositions may comprise one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a concentrated composition as taught inExample 1 may be used. For example, a microorganism-containingcomposition may comprise IN-M1, Accession No. PTA-12383. For example, amicroorganism-containing composition may comprise IN-M1, Accession No.PTA-12383 and one or hydrocarbon degrading microorganisms. Methods ofusing a microorganism-containing composition comprise applying aneffective amount of a microorganism-containing composition to a site orcontainer comprising oil or other hydrocarbons and water and/or soil andreducing or eliminating at least a portion of the oil or otherhydrocarbons in the water and/or soil.

The present invention comprises compositions and methods useful intreatments for wildlife or other animals that may be affected bypollutants or contaminants. For example, and not to be limiting, thecompositions and methods comprise treating birds or other animals orwildlife contaminated by oil or hydrocarbons, thereby reducing theamount of time and handling needed to clean the oil or hydrocarbons fromthe animals. In one aspect, a microorganism-containing composition maybe diluted and grown to provide large quantities of a ready-to usecomposition in about 5-10 days or a microorganism-containing compositionmay be used in a concentrated form. Methods of using the compositionsdisclosed herein in the treatments for animals comprise 1) optionally,diluting the composition, and, spraying a diluted solution onto the coator feathers of the animal, 2) leaving the compositions on the coat orfeathers of the animal for a pre-determined time period such as fromminutes to hours, and 3) rinsing the animal or allowing the animal tobathe in a pool of water, or contacting the animal with a composition,comprising an even more diluted concentration of themicroorganism-containing composition. This method can be repeated. Inone aspect, a shower spray of the compositions can be used to assist theremoval of the oil or hydrocarbon residues. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillusoryzae, Candida utilis, Streptococcus lactis, Lactobacillis planterum,Lactobacillis helveticus and Lactobacillus caseii. For example, avariation of a concentrated composition as taught in Example 1 may beused. Microorganism-containing compositions may comprise one or moremixed cultures, for example, IN-M1, or compositions comprising IN-LH1,IN-BS1, IN-SC1, and/or IN-RP1, in combination or individually, and/orwith other microorganisms. For example, a concentrated composition astaught in Example 1 may be used. For example, a microorganism-containingcomposition may comprise IN-M1, Accession No. PTA-12383. For example, amicroorganism-containing composition may comprise IN-M1, Accession No.PTA-12383 and one or hydrocarbon degrading microorganisms. Methods ofusing a microorganism-containing composition comprise applying aneffective amount of a microorganism-containing composition to an animaland reducing or eliminating at least a portion of the contaminant, suchas oil or other hydrocarbons.

The present invention comprises compositions and methods useful in thebioremediation of soil. In one aspect, the compositions and methods mayincrease the water retention of the soil. In one aspect, thecompositions and methods may stimulate the root growth of plants livingon or in the soil. In one aspect, the compositions and methods mayreduce the risk of infestation by insects. In one aspect, thecompositions and methods may reduce the risk of disease. In one aspect,the compositions may aerate the soil, degrade chemical or organiccontaminants, and help the plants to take up the organic nutrients thatare naturally present or added in the soil. For example, and not to belimiting, the compositions and methods of the present invention areuseful in the elimination of brown patches and areas of dead grass ongolf course putting greens. In one aspect, the compositions and methodscan reduce the need to irrigate soil in order to maintain healthy plantgrowth. Compositions can be a concentrate and can either be used as aconcentrate, or diluted 100:1 to 1:300 in water, and then can be sprayedon the surface of soil requiring treatment. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis plantarum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. Microorganism-containing compositions may comprise one ormore mixed cultures, for example, IN-M1, or compositions comprisingIN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination or individually,and/or with other microorganisms. For example, amicroorganism-containing composition may comprise IN-M1,

Accession No. PTA-12383. For example, a microorganism-containingcomposition may comprise IN-M1, Accession No. PTA-12383 and one ormicroorganisms, such as microorganisms derived from the treatment siteor a similar site. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site such as all or a portionof a golf course, wherein the retention and maintenance of the grass ofthe golf course is improved and the transition from chemical andsynthetic molecules to sustainable organic maintenance is assisted bysuch methods.

Bioremediation of soil may also comprise providing amicroorganism-containing composition to plants or rhizospheres that maybe added to a site or providing a microorganism-containing compositionto plants or rhizospheres that are present in a site. For example, at acontaminated or polluted site, plants may be planted in the contaminatedor polluted site wherein the plants have had a microorganism-containingcomposition applied to the roots. A combined composition of amicroorganism-containing composition, comprising one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a concentrated composition as taught inExample 1 may be used. For example, a microorganism-containingcomposition may comprise IN-M1, Accession No. PTA-12383 combined withorganisms derived from the site or a similarly contaminated site may beprovided to the plants. The plants are allowed to grow and may beharvested or removed after a certain time period, to remove contaminantsfrom the site without removing the soil itself The plants may eventuallybe used as a biomass fuel source. For example, the plants at acontaminated site may be contacted by a microorganism-containingcomposition, such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms. Forexample, a concentrated composition as taught in Example 1 may be used.For example, a microorganism-containing composition may comprise IN-M1,Accession No. PTA-12383 and optionally, the soil may be contacted also.A combined composition of a microorganism-containing composition, suchas a microorganism-containing composition comprising one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a concentrated composition as taught inExample 1 may be used. For example, a microorganism-containingcomposition may comprise IN-M1, Accession No. PTA-12383 combined withorganisms derived from the site or a similar site may be provided to theplants.

Though not wishing to be bound by any particular theory, it is thoughtthat a microorganism-containing composition of the present inventionstimulates the formation of a beneficial microfilm on the rhizosphere ofplants that allows for interactions between organisms in the soil andthe plant. This enhanced interaction allows for better transfer ofnutrients into the plants some of which may include metabolites and/orelements derived from the contamination, which results in rapidmaturation, flowering, more branching and increased yield of fruitingbodies. Use of a microorganism-containing composition, such as one ormore mixed cultures, for example, IN-M1, or compositions comprisingIN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination or individually,and/or with other microorganisms have shown stimulation of plant growthfor plants grown in organic material with mychorrhyzal fungi; algaeblooms in nutrient solutions were suppressed; suppression of commonplant infections; and suppression of algae growth on turf grasses ofgolf greens and mold on leaves of growing plants.

Compositions and methods of the present invention comprise enhancementof seed germination. Providing a microorganism-containing composition,such as one or more mixed cultures, for example, IN-M1, or compositionscomprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination orindividually, and/or with other microorganisms to seeds of lettuces andother microgreens showed that seed germination was decreased by 50%. Acomposition comprising IN-M1 at a concentration of 1×10⁶ cells/mL wassprayed onto lettuce and similar greens seeds. The seeds were germinatedhydroponically on an organic matrix and wet weight and shoot height wasan average of 20% greater than the same seeds not treated with thecomposition. Seeds so treated resulted in plants that had a 20% increasein biomass in 7 days, compared to plants in the seeds were not treated.

Seed germination in turf grasses, such as that used in golf courses, wasenhanced by treating the turf grass seeds with amicroorganism-containing composition , such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms. For example, a concentrated composition as taught inExample 1 may be used. For example, a microorganism-containingcomposition comprising IN-M1, Accession No. PTA-12383 at a concentrationof 1×10⁶ cells per mL was sprayed on a green where the seed had beenscattered on the putting greens of a golf course in the southern US. TheKentucky blue grass was established during the time the summer grass wasdormant. The Kentucky Blue grass grew faster, with germinationaccelerated from 7 days to 5 days and became established at a ratefaster than grass where the seeds were not sprayed, in 3 weeks ascompared to untreated seeds of 4 weeks.

The present invention comprises compositions and methods useful in thetreatment or management of commercial and residential septic systems.Compositions and methods are useful the reduction of sludge from sewersor septic systems, thereby reducing health risks from infections andtoxic substances including hydrogen sulfide, ammonia and volatileorganic carbons. Compositions and methods are useful in controlling theodors from commercial or residential septic systems. In one aspect,compositions are used to treat biosolids or sludge contained in pipes.In one aspect, methods and compositions can be used to treat biosolidsor sludge removed from sewers and lift stations which may be containedat storage facilities. In one aspect, methods and compositions areuseful in the remediation of septic seepage or drain fields. In oneaspect, compositions and methods can be used to remove fat, oil, andgrease (FOG) from wastewater. In one aspect, the compositions andmethods can be used to treat water polluted by manure from industrialfarms. The compositions can be concentrated (see Example 1) and caneither be used as concentrated, or diluted 100:1 to 1:10000 in water,and then can be sprayed on the seepage field or area requiringremediation. Compositions may comprise a microorganism-containingcomposition comprising Rhodopseudomonas palustris, Bacillus subtilis,Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae,Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus and Lactobacillus caseii. For example, aconcentrated composition as taught in Example 1 may be used. Forexample, compositions such as microorganism-containing compositions maycomprise one or more mixed cultures, for example, IN-M1, or compositionscomprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination orindividually, and/or with other microorganisms. For example, aconcentrated composition as taught in Example 1 may be used. Forexample, a microorganism-containing composition may comprise IN-M1,Accession No. PTA-12383 may be used. Methods of using amicroorganism-containing composition comprise applying an effectiveamount of a microorganism-containing composition to a site or containercomprising sludge, allowing the composition to remain for a time period,for example time to grow and reproduce at the treated site or surface,and reducing or eliminating at least a portion of the sludge. Methods ofusing a microorganism-containing composition comprise applying aneffective amount of a microorganism-containing composition to a site orcontainer comprising Fat Oil and Grease (FOG), allowing the compositionto remain for a time period, for example time to grow and reproduce atthe treated site or surface, and reducing or eliminating at least aportion of the FOG. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to holding tank of industrial farmwastes, allowing the composition to remain for a time period, forexample time to grow and reproduce at the treated site or surface, andreducing or eliminating at least a portion of the the wastes.Compositions maybe delivered using existing equipment, and interfacingwith existing and new aeration, mixing separation/filtration and,monitoring systems useful in the treatment or management of commercialand residential septic systems.

Applying, as used herein, may be contacting the material with amicroorganism-containing composition as taught herein.

The present invention comprises compositions and methods useful in thetreatment or management of municipal or industrial wastewater treatment.In one aspect, the compositions and methods can reduce the carbondioxide, methane, ammonia hydrogen sulfide and other greenhouse gasemissions and odour causing compounds. In one aspect, the compositionsand methods may reduce biofilm causing bacteria and ecological upsetslike filamentous bacteria in sewer lines, pumping/lift stations and inthe waste water treatment plant. In one aspect, the compositions andmethods can reduce the biological oxygen demand and chemical oxygendemand in polluted water. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis plantarum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as microorganism-containingcompositions, such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms. Forexample, a concentrated composition as taught in Example 1 may be used.For example, a microorganism-containing composition may comprise IN-M1,Accession No. PTA-12383 may be used. Methods of using amicroorganism-containing composition comprise applying an effectiveamount of a microorganism-containing composition to a municipal orindustrial wastewater system, allowing the composition to remain for atime period, for example time to grow and reproduce at the treated siteor surface, and reducing or eliminating at least a portion of thewastes. Compositions maybe delivered using existing or new equipment,such as existing and new aeration systems, mixing and monitoring systemsand biofilters for municipal or industrial wastewater treatment.

The present invention comprises compositions and methods useful in thetreatment and/or recycling of water used in industrial processes.Compositions may comprise a microorganism-containing compositioncomprising Rhodopseudomonas palustris, Bacillus subtilis, Pseudomonasaeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae, Candidautilis, Streptococcus lactis, Lactobacillis plantarum, Lactobacillishelveticus and Lactobacillus caseii. For example, a concentratedcomposition as taught in Example 1 may be used. For example,compositions such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused.

Methods of using a microorganism-containing composition compriseapplying an effective amount of a microorganism-containing compositionto a site or container comprising wastes from industrial processes,allowing the composition to remain for a time period, for example timeto grow and reproduce at the treated site or surface, allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and reducing or eliminating atleast a portion of the wastes. For example, compositions and methods areuseful in the treatment of animal waste generated at dairy farms in theproduction of milk. The compositions and methods can reduce thegreenhouse gas emissions like carbon dioxide, methane, as well asammonia and other odor causing compounds in the facility, the lagoonsand in the sludge or resulting compost which may be spread on soil as ahealthy soil amendment. Compositions and methods may improve the qualityand performance of the treated biosolids as a soil amendment.Compositions may be delivered using existing equipment, existing andinterfacing with new aeration, mixing and monitoring systems formunicipal or industrial wastewater treatment. Compositions may beincluded in anaerobic and aerobic protocols, interfacing with existingtechnology and included in together with aeration, mixing and monitoringsystems as separate solutions and/or systems industrial waste waterrecycling.

The present invention comprises compositions and methods useful in thetreatment of water contained by dams. In one aspect, compositions andmethods are used to treat water contained by a dam for the purposes ofgenerating hydroelectric power. For example, and not to be limiting, thetreatment of dammed water can include reducing the output of carbondioxide from the water source. Compositions may accelerate thedegradation of vegetation buried under the water thus reducinggreenhouse gas emissions. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis plantarum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container comprisingstagnant or contained water, allowing the composition to remain for atime period, for example time to grow and reproduce at the treated siteor surface, and reducing or eliminating a portion of the greenhouse gasor carbon dioxide released or present.

The present invention comprises compositions and methods useful incontrolling odors generated by composting facilities. The compositionsand methods can reduce the carbon dioxide, methane, and other greenhousegas emissions, as well as reducing ammonia and other odor causingcompounds. The compositions and methods may accelerate the compostingprocess in municipal and industrial scale composting facilities. Thecompositions and methods may decrease heavy metal levels, when presentand improve the final product quality and performance as a healthy soilamendment. For example, compositions such as one or more mixed cultures,for example, IN-M1, or compositions comprising IN-LH1, IN-BS1, IN-SC1,and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container comprisingodiferous material and reducing or eliminating at least a portion of themalodors released by the site or container.

The compositions and methods can reduce the corrosive nature of theleachate, render the leachate beneficial to the process and reduce odorsthereof. The compositions and methods can interact with biofilterinstallations augmenting the performance thereof. Compositions maycomprise a microorganism-containing composition comprisingRhodopseudomonas palustris, Bacillus subtilis, Pseudomonas aeruginosa,Saccharomyces cerevisiae, Aspergillus oryzae, Candida utilis,Streptococcus lactis, Lactobacillis plantarum, Lactobacillis helveticusand Lactobacillus caseii, for example, a concentrated composition astaught in Example 1. For example, compositions such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container comprisingleachate, allowing the composition to remain for a time period, forexample time to grow and reproduce at the treated site or surface, andreducing at least a portion of the the corrosive nature of the leachate,rendering at least a portion of the the leachate beneficial to theprocess. The compositions may sequester all or some of the leachate ormay alter or modify the leachate to reduce its toxicity.

Compositions of the present invention may be delivered using existingequipment, interfacing with existing and new separated organic wastemanagement facilities including, windrows, static piles and in vesselfacilities, anaerobic and aerobic protocols, interfacing with existingequipment and with new equipment or systems for aeration, grinding,mixing, biofilters and monitoring systems as separate solutions and/orsystems for municipal or industrial scale composting facilities.

The present invention comprises compositions and methods useful in thedecontamination of soil and water that are contaminated as a result ofnatural disasters. For example, and not to be limiting, a naturaldisaster can be a flood, an earthquake, a hurricane, a tsunami or anyother natural disaster. In one aspect, compositions and methods can beused to suppress at least a portion of the pathogens that enter thewater system following a natural disaster. In one aspect, thecompositions and methods can be used to remediate water contaminated byVibrio cholerae. The compositions of the present invention can bedelivered into the water system requiring remediation by providing amicroorganism-containing composition directly to the water, or by useexisting equipment or with known delivery systems. Water can be treatedin existing locations such as holding tanks, lagoons or directly at thesource in latrines, gutters, or stagnant groundwater. Deliverymechanisms can be optimized based on different circumstances.Compositions may comprise a microorganism-containing compositioncomprising Rhodopseudomonas palustris, Bacillus subtilis, Pseudomonasaeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae, Candidautilis, Streptococcus lactis, Lactobacillis plantarum, Lactobacillishelveticus and Lactobacillus caseii. For example, a concentratedcomposition as taught in Example 1 may be used. For example,compositions such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused. Compositions may comprise natural antibiotic and anti parasiticorganisms targeting disease affecting displaced populations andpopulations plagued by poor sanitation. Methods of using amicroorganism-containing composition comprise applying an effectiveamount of a microorganism-containing composition to a site or containerof water comprising unwanted organisms such as Vibrio cholera, allowingthe composition to remain for a time period, for example time to growand reproduce at the treated site or surface, and reducing oreliminating at least a portion of the microorganisms such as Vibriocholerae.

The present invention comprises compositions and methods useful in thecreation and maintenance of healthy soil environments, thereby allowingfor healthy plant growth. Storm or water run-off is treated inengineered reservoirs, or other locations such as on a farm or in anindustrial complex, before the run-off water reaches the surface wateror contaminates the ground, the run-off is treated with a composition ofthe present invention. Therefore treating at source before contaminationis transported from contaminated site to other sites aids in thecreation and maintenance of healthy soil environments. Such treatmentsmay encourage vegetation in the watershed area that will aid in treatingthe run-off. In one aspect, compositions and methods can be used toameliorate the contaminating effects of water runoff or storm waterrunoff by treating at source contamination such as manure pits and golfcourses. In one aspect, compositions and methods can be used toameliorate the contaminating effects of water runoff or storm waterrunoff by treating porous hard surfaces to clear the pores of thesurface to clear organic debris and allow penetration of the storm waterthrough the pores in pervious concrete.

Compositions may comprise a microorganism-containing compositioncomprising Rhodopseudomonas palustris, Bacillus subtilis, Pseudomonasaeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae, Candidautilis, Streptococcus lactis, Lactobacillis planterum, Lactobacillishelveticus and Lactobacillus caseii. For example, a concentratedcomposition as taught in Example 1 may be used. For example,compositions such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused. Methods of using a microorganism-containing composition compriseapplying an effective amount of a microorganism-containing compositionto a site or container contacted by stormwater runoff, allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and reducing or eliminating atleast a portion of the contaminants carried to the site or container inthe water runoff.

The present invention comprises compositions and methods useful inhydroponic systems for plant growth. Methods of the present inventioncomprise contacting the root systems of plants grown in hydroponicconditions, or spraying the leaves, stems, fruits or roots of plantsgrown in a hydroponic system. A dilute concentration, such as 1×10³ to5×10⁴ cells per mL, may be added to the circulating media in ahydroponic system to reduce algae bloom formation and optionally totreat root systems of plants in the circulating media.

Compositions used in such hydroponic systems may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis planterum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to plants or containers of theplants or providing a composition to the circulating media or othermedia used in hydroponic growing conditions, allowing the composition toremain for a time period, for example time to grow and reproduce at thetreated site or surface, and enhancing the growth of the plants in thehydroponic system, and optionally reducing the amount of algae or otherunwanted growth in the media, or on containers or piping.

The present invention comprises compositions and methods useful in theremediation of mold growing in an area following or in damp or humidenvironments. In one aspect, the compositions and methods can be used totreat and prevent the growth of mold in any building that has sustainedwater damage as a result of flooding. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis plantarum, Lactobacillis helveticus and Lactobacilluscaseii. For example, compositions such as microorganism-containingcompositions, such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused. Methods of using a microorganism-containing composition compriseapplying an effective amount of a microorganism-containing compositionto a site or container comprising mold growth and reducing oreliminating at least a portion of the mold growth.

The present invention comprises compositions and methods useful inimproving the qualities of soil. In one aspect, compositions and methodsmay increase the water retention of the soil. In one aspect,compositions and methods may stimulate the root growth of plants livingon or in the soil. In one aspect, compositions and methods may reducethe risk of infestation by insects. In one aspect, compositions andmethods may reduce the risk of disease. In one aspect, compositions mayaerate the soil, degrade chemical or organic contaminants, and help theplants to take up the organic nutrients that are naturally present oradded in the soil. Therefore, compositions and methods can be used toenhance the growth of lawns, flowers, fruits, vegetables, orchards orvineyards, trees or forests. In one aspect, compositions and methods canbe used in greenhouses. In one aspect, compositions and methods can beused in municipal and urban plantations in difficult environments. Inone aspect, compositions and methods can be used at the side of roadswhere soil is contaminated, for example, with salt and oil. In oneaspect, compositions and methods can be used for example, on militaryfacilities where soil may be contaminated with oil, salt, or heavymetals. Compositions may comprise a microorganism-containing compositioncomprising Rhodopseudomonas palustris, Bacillus subtilis, Pseudomonasaeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae, Candidautilis, Streptococcus lactis, Lactobacillis planterum, Lactobacillishelveticus and Lactobacillus caseii. For example, a concentratedcomposition as taught in Example 1 may be used. For example,compositions such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused. Methods of using a microorganism-containing composition compriseapplying an effective amount of a microorganism-containing compositionto a site or container comprising plants, allowing the composition toremain for a time period, for example time to grow and reproduce at thetreated site or surface, and enhancing the growth of the plants. Methodsof using a microorganism-containing composition comprise applying aneffective amount of a microorganism-containing composition to a site orcontainer comprising soil or compost or a mixture thereof, allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and improving the soilquality, such as water retention, increased microbial concentration, orother beneficial qualities.

The present invention comprises compositions and methods useful in thecontrol of insects, by the microorganisms of the composition colonizingor being present on the leaf surface and around the plant roots,creating a barrier against harmful insects and fungal spores. Thecompositions and methods may be used to suppress for example mildew andinsect infestations whereby the beneficial microbial species colonizeplants and soil. In one aspect, the compositions boost the immune systemof the plant to fight infection. In one aspect, compositions of thepresent invention may act as a delivery system for active agents such asbiological insecticides or larvecides as such as bti, as well as naturalingredients. It is theorized that providing a composition of the presentinvention provides competitive inhibition for pathogenic organisms bythe microorganisms in the composition. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis planterum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as microorganism-containingcompositions, such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused. Methods of using a microorganism-containing composition compriseapplying an effective amount of a microorganism-containing compositionto a site or container comprising plants, allowing the composition toremain for a time period, for example time to grow and reproduce at thetreated site or surface, and reducing or eliminating at least a portionof the pathological effects on the plants, or aid in delivery of desiredactive agents, such as insecticides, larvecides or other desired activeagents. Methods of application include, but are not limited to, sprayingfoliage, soil and fruit.

The present invention comprises compositions and methods useful in beekeeping. In one aspect, the compositions and methods can be used tocontrol varroa mites inside bee hives. In one aspect the hives aresprayed inside with a microorganism-containing composition at 5×10³ to5×10⁴cells per mL in water. In another aspect a microorganism-containingcomposition can be used to clean and wipe down the hives. It istheorized that providing a composition of the present invention providescompetitive inhibition for pathogenic organisms by the beneficialmicroorganisms in the composition. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcuslactis,iLactobacillis plantarum, Lactobacillis helveticus andLactobacillus caseii. For example, a concentrated composition as taughtin Example 1 may be used. For example, compositions such asmicroorganism-containing compositions, such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container comprisinginsects to remove harmful organisms.

The present invention comprises compositions and methods useful in thetechniques of hydroponic or aeroponic agriculture, or in verticalfarming methods. Compositions of the present invention can be added tothe nutrient solution of the plants to increase the uptake of nutrientsby the plants. Compositions may also be sprayed on the plant surfacesand in the air to prevent pathogenic organisms from colonizing leavesand fruiting bodies or growing on the plant Other surfaces andsurroundings of plants, including but not limited to containers,shelves, greenhouses, rock wool, growing media surfaces, may be providedcompositions of the present invention. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis planterum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as microorganism-containingcompositions, such as one or more mixed cultures, for example, IN-M1, orcompositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, incombination or individually, and/or with other microorganisms may beused. Methods may comprise providing a microorganism-containingcomposition to all or a portion of a plant or seeds, allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and enhancing the biologicalactivities of the plant or seeds, such as increasing germination rate,increasing biomass of the plant, reducing or modulating infection of theplant or seeds, increasing fruit setting rate and flowering, increasingtuber development, increasing the length of production time for theplants, increasing the productive lifespan of the treated plant,increasing nutrient uptake, and treating the plants and seeds, andfluids or surfaces contacted by the plant or seeds, such as media orcontainers, to reduce a portion of the pathogenic or endogenous microbespresent.

The present invention comprises compositions and methods useful inaquaculture or pisciculture. In one aspect a microorganism-containingcomposition is used to suppress odors. In another aspect amicroorganism-containing composition is used to degrade fish waste,ammonia and suppresses algae. In another aspect amicroorganism-containing composition suppresses infection and isbeneficial for fish coats and digestion. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis planterum, Lactobacillis helveticus and Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container comprisingfish or fish wastes, or waters in which fish are kept, allowing thecomposition to remain for a time period, for example time to grow andreproduce at the treated site or surface, and reducing the odors orunwanted algal growth, or aiding in the growth of the fish.

The present invention comprises compositions and methods useful in thedecontamination of industrial by-products from and used in theconstruction industry. In one aspect, a microorganism-containingcomposition of the present invention can be used as an additive toconcrete or added to cement during the cement-making process to increasepermeability.

The present invention comprises compositions and methods useful in thehealthcare industry. For example, and not to be limiting, thecompositions and methods can be used to enhance air quality, suppresspathogens, clean and sanitize healthcare facilities or remediate toxicwaste generated by healthcare facilities. Additionally, compositions ofthe present invention may be to aid in human or animal health. Forexample, the compositions may be used as source material for theextraction or purification of active agents that are useful as foodsupplements, in cosmetics or are nutriceutical cosmeceticals, probioticor pharmaceutical compositions such as antioxidants and biologicalsubstances and compounds that aid digestive processes andgastrointestinal disease. Compositions may be added to foods orcosmetics, or may function as supplements separately from foods.Compositions may comprise factors isolated from or amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis plantarum, Lactobacillis helveticus and/or Lactobacilluscaseii. For example, a concentrated composition as taught in Example 1may be used. For example, compositions such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container, allowingthe composition to remain for a time period, for example time to growand reproduce at the treated site or surface, and at least a portion ofthe pathogenic organisms are reduced or eliminated. A composition of thepresent invention may provide competitive inhibition by colonizing asurface and preventing the colonization of pathogenic bacteria, such asMRSA. Compositions of the present invention may be used in treatmentmethods of biological, chemotoxic (including urine and other biologicalwaste from cancer patients and other patients) and radioactive wasteincluding radioactive isotope tracers, treatments and implant patientsat source, and comprise providing a microorganism-containing compositionto a site to be treated, allowing the composition to remain for a timeperiod, for example time to grow and reproduce at the treated site orsurface, and reducing or eliminating at least a portion of the waste orunwanted materials or pathogens.

The present invention comprises compositions and methods useful in thetransportation industry. For example, and not to be limiting, thecompositions and methods can be used to clean or treat diesel spillsform engines trucks, trains or aircraft. In one aspect,microorganism-containing compositions and methods can be used to treatwastewater generated on passenger buses, trains or aircraft.Compositions of the present invention may be used in treatment methodsfor wastes from latrines, portapotties, oily water, such as bilge water,waste water from toilet facilities, restaurant and kitchens ontransportation vehicles, vessels and the like. For example, compositionssuch as one or more mixed cultures, for example, IN-M1, or compositionscomprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, in combination orindividually, and/or with other microorganisms may be used.

The present invention comprises compositions and methods useful in theraising or keeping of animals. For example, and not to be limiting, theanimals can be raised or kept as pets or as farm animals and can bedogs, cats, horses, cows, pigs or fish. In one aspect, the compositionsand methods can be used to control animal odor. In one aspect, this canbe accomplished by spraying the compositions on the animal's bedding ordirectly on the animal. In one aspect, animals can ingest thecompositions of the present invention with their food or water, therebyimproving their health, digestion and odor. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis plantarum, Lactobacillis helveticus and Lactobacilluscaseii.

For example, a concentrated composition as taught in Example 1 may beused. For example, compositions such as one or more mixed cultures, forexample, IN-M1, or compositions comprising IN-LH1, IN-BS1, IN-SC1,and/or IN-RP1, in combination or individually, and/or with othermicroorganisms may be used. Methods of using a microorganism-containingcomposition comprise applying an effective amount of amicroorganism-containing composition to a site or container which iscontacted by an animal and reducing or eliminating at least a portion ofthe odors, or the pathogenic organisms. Methods may comprise providing amicroorganism-containing composition as an animal feed.

The present invention comprises compositions useful as additives inproducts for human consumption or use. For example, and not to belimiting, the compositions can be used as additives in probiotic healthdrinks and food supplements alcoholic or non-alcoholic fermentedbeverages, fermented foods, salad dressings, marinades, culinary spicesor supplements, dietary supplements, shampoos, soaps, anti-fungalmedications, toothpaste or mouthwash. Compositions may comprise amicroorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis planterum, Lactobacillis helveticus or Lactobacilluscaseii, in combination, or individually. For example, a concentratedcomposition as taught in Example 1 may be used. For example,compositions such as microorganism-containing compositions, such as oneor more mixed cultures, for example, IN-M1, or compositions comprisingIN-LH1, IN-BS1, IN-SC1, and/or IN-RP1, or a microorganism-containingcomposition comprising Rhodopseudomonas palustris, Bacillus subtilis,Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae,Candida utilis, Streptococcus lactis, Lactobacillis planterum,Lactobacillis helveticus or Lactobacillus caseii, in combination orindividually, and/or with other microorganisms may be used. Methods ofusing a microorganism-containing composition comprise adding aneffective amount of a microorganism-containing composition to afoodstuff, cosmetic agent, cleansing agent or providing a composition asan ingestible composition. Foodgrade products may be fermented with themicroorganisms.

The present invention comprises compositions that can be ingested orapplied as medications. For example, and not to be limiting,microorganism-containing compositions can be used as treatmentcompositions for skin or hair disorders, or digestive ailments. Forexample, compositions such as one or more mixed cultures, for example,IN-M1, or compositions comprising IN-LH1, IN-BS1, IN-SC1, and/or IN-RP1,or a microorganism-containing composition comprising Rhodopseudomonaspalustris, Bacillus subtilis, Pseudomonas aeruginosa, Saccharomycescerevisiae, Aspergillus oryzae, Candida utilis, Streptococcus lactis,Lactobacillis planterum, Lactobacillis helveticus or Lactobacilluscaseii, in combination or individually, and/or with other microorganismsmay be used.

The present invention comprises compositions and methods useful indomestic settings such as a household.

Methods and compositions disclosed herein may comprise use ofmicroorganisms that are from treatment sites, such as biologicalsurfaces of plants or animals, or environmental sites such ascontaminated or polluted sites, and such microorganisms are added to oradmixed with a microorganism-containing composition of the presentinvention. Methods comprise adding microorganisms isolated from atreatment site to compositions comprising one or more mixed cultures,for example, IN-M1, or compositions comprising IN-LH1, IN-BS1, IN-SC1,and/or IN-RP1, or Rhodopseudomonas palustris, Bacillus subtilis,Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae,Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus or Lactobacillus caseii, in combination orindividually, and/or with other microorganisms to form combinedcompositions. Such combined compositions may be used at the originatingsites to remove contaminants, or other desired treatments. This issometimes referred to as in situ remediation, which utilizes theindigenous, contaminant-degrading microorganisms which are present atthe contaminated site. These organisms are present at the site and arecapable of some kind of degradation of an element of the site.Generally, when unaltered and in its original state, the indigenousmicroorganisms' decontaminating action proceeds at too low a level andtoo slow a rate to effectively decontaminate the area. Additionally, theorganisms may be only able to chemically change one contaminant in amixed collection of pollutants, or only make a few chemical changes in achemically-complex contaminating molecule. The growth and activities ofthe naturally occurring microorganisms are enhanced by forming acombined composition comprising a microorganism-containing compositionof the present invention. A method for making a combined compositioncomprises feeding and growing a small amount of organisms from thecontaminated soil or water site and admixing it with amicroorganism-containing composition to stimulate growth of a naturalbeneficial microbial remediation community or microorganisms. Thiscombined composition of the present invention culture may be thenreintroduced into the contaminated environment or used in other similarsituations. For example, compositions such as one or more mixedcultures, for example, IN-M1, or compositions comprising IN-LH1, IN-BS1,IN-SC1, and/or IN-RP1, or Rhodopseudomonas palustris, Bacillus subtilis,Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus oryzae,Candida utilis, Streptococcus lactis, Lactobacillis plantarum,Lactobacillis helveticus or Lactobacillus caseii, in combination orindividually, and/or with other microorganisms may be combined withorganisms from a polluted or contaminated site, and the combinedcomposition may be grown in a container or may be added to thecontaminated or polluted site, or sites similar to the original site tomodulate, such as enhance, the breakdown occurring at the site.

It will be understood that the aspects of the present invention whichhave been described are illustrative of some of the applications of theprinciples of the present invention. Numerous modifications may be madeby those skilled in the art without departing from the true spirit andscope of the invention. The terminology used herein is for the purposeof describing particular aspects only and is not intended to belimiting.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

The term “treating” refers to inhibiting, preventing, curing, reversing,attenuating, alleviating, minimizing, suppressing or halting thedeleterious effects of a condition, contaminant, toxicant, pollutantand/or causing the reduction, remission, or regression of a condition.Those of skill in the art will understand that various methodologies andassays can be used to assess the development of a condition, andsimilarly, various methodologies and assays may be used to assess thereduction, remission or regression of the condition.

As used herein, the term “determining” can refer to measuring orascertaining a quantity or an amount or a change in activity. Forexample, determining the characteristics of microorganisms or growingcultures as used herein can refer to the steps that the skilled personwould take to measure or ascertain some quantifiable value in a sample.The art is familiar with the ways to measure characteristics in asample. The term sample is used in its common meaning of a portion froma larger solution, from a site, from a culture, or other larger entityfrom which a portion, the sample, can be removed and optionally actedupon.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

The invention has been described with reference to specific embodimentsthereof. It will, however, be evident that various modifications andchanges may be made thereto without departing from the broader spiritand scope of the invention. The specification and drawings are,accordingly, to be regarded in an illustrative rather than a restrictivesense.

The present invention comprises microorganism-containing compositionsand methods for making and using such compositions. For example, amicroorganism-containing composition comprises a composition comprisingisolated bacteria, at least one isolated yeast, and at least oneisolated micorrhyzal fungus, wherein the bacteria comprise at least alactobacillus, and at least a bacillus, wherein the bacteria wereselected based on enzyme profiles so that the bacteria arecomplementary. The Lactobacillus may be L. helveticus, or theLactobacillus may be L.helveticus, IN-LH1, Accession No. PTA-12386. TheBacillus may be B. subtilis, or the Bacillus may be B. subtilis, IN-BS1,Accession No. PTA-12385. The yeast may be Saccharomyces cerevisiae, orthe yeast may be Saccharomyces cerevisiae, referred to as IN-SC1,Accession No. PTA-12384. The microorganism-containing composition mayfurther comprise an isolated photosynthetic bacteria. The photosyntheticbacteria may be Rhodopseudomonas palustris, or the photosyntheticbacteria is Rhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387.The microorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method using a composition of the present invention comprises a methodof bioremediation of a site, comprising, providing amicroorganism-containing composition to a site comprising at least onecontaminant, wherein the microorganism-containing composition comprisesat least one isolated bacteria, at least one isolated yeast, and atleast one isolated micorrhyzal fungus, wherein the bacteria comprise atleast a lactobacillus and at least a bacillus, wherein the bacteria wereselected based on enzyme profiles so that the bacteria arecomplementary, allowing the microorganisms of themicroorganism-containing composition to grow and reproduce for a periodof time; and reducing at least a portion of at least one contaminant atthe site. The Lactobacillus may be L. helveticus, or the Lactobacillusmay be L.helveticus, IN-LH1, Accession No. PTA-12386. The Bacillus maybe B. subtilis, or the Bacillus may be B. subtilis, IN-BS1, AccessionNo. PTA-12385. The yeast may be Saccharomyces cerevisiae, or the yeastmay be Saccharomyces cerevisiae, referred to as IN-SC1, Accession No.PTA-12384. The microorganism-containing composition may further comprisean isolated photosynthetic bacteria. The photosynthetic bacteria may beRhodopseudomonas palustris, or the photosynthetic bacteria isRhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387. Themicroorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein. The method may further comprise, at apredetermined time after providing the microorganism-containingcomposition, measuring the level or amount of at least one contaminantat the site and comparing it to a measurement of the level or amount ofat least one contaminant at the site obtained prior to providing themicroorganism-containing composition. The method may further comprise ata predetermined time after providing the microorganism-containingcomposition, assessing at least one characteristic of the condition ofthe contaminated site and comparing it to that characteristic of thecondition of the site prior to providing the microorganism-containingcomposition. Assessing at least one characteristic of the condition ofthe site may comprise measuring at least one characteristic of the site,wherein a characteristic comprises the color, the smell, thecleanliness, the amount of plant growth, the amount of microbial growth,the amount of animal growth, the amount of biosolids, at the site andcomparing that characteristic measurement to a measurement of thatcharacteristic of the condition of the site taken prior to providing themicroorganism-containing composition, to provide a measurement of achange in the condition of the site. The site may comprise a mine, amunicipal water system, a contaminated site, a polluted site, a septicsystem, a golf course, lentic water, a lake, a pond, a contained body ofwater, lotic water, a flowing stream, a moving body of water, acirculating contained body of water, leachate from a landfill, a minetailing, industrial waste water or soil, a contained body of soil,contained material removed from a contaminated site, water or soilbehind a dam, water or soil from a farm or facility with animals, wateror soil from a farm or a facility for plants, water or soil from a wastetreatment plant, such as a landfill, municipal water and waste treatmentsystem, commercial water and waste treatment system, industrial waterand waste treatment system, or private water and waste treatment system.The method may further comprise, prior to adding themicroorganism-containing composition to the site, obtaining a samplefrom the site and adding at least a portion of the sample to themicroorganism-containing composition to form a combinedmicroorganism-containing composition, and allowing microorganisms of thecombined microorganism-containing composition to grow and reproduce. Themethod may further comprise providing the combinedmicroorganism-containing composition comprising one or moremicroorganisms from the site to the site.

A method using a composition of the present invention comprises a methodof affecting, enhancing, modulating, stimulating or aiding plant growth,comprising, providing a microorganism-containing composition to a sitewhere plants are grown, to a plant or to a plant at a particular growthstage, or combinations thereof, wherein the microorganism-containingcomprises at least one isolated bacteria, at least one isolated yeast,and at least one isolated micorrhyzal fungus, wherein the bacteriacomprise at least a lactobacillus and at least a bacillus, wherein thebacteria were selected based on enzyme profiles so that the bacteria arecomplementary; and allowing microorganisms of themicroorganism-containing composition to grow and reproduce for a periodof time. The Lactobacillus may be L. helveticus, or the Lactobacillusmay be L.helveticus, IN-LH1, Accession No. PTA-12386. The Bacillus maybe B. Subtilis, or the Bacillus may be B. subtilis, IN-BS1, AccessionNo. PTA-12385. The yeast may be Saccharomyces cerevisiae, or the yeastmay be Saccharomyces cerevisiae, referred to as IN-SC1, Accession No.PTA-12384. The microorganism-containing composition may further comprisean isolated photosynthetic bacteria. The photosynthetic bacteria may beRhodopseudomonas palustris, or the photosynthetic bacteria isRhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387. Themicroorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein. The method may comprise applying themicroorganism-containing composition to the seeds of plants. The methodmay comprise applying the microorganism-containing composition to theroots of plants. The method may comprise applying themicroorganism-containing composition to the leaves and stalks of plants.The method may comprise enhancing seed germination. The method maycomprise enhancing root production. The method may comprise increasingthe per plant yield of treated plants compared to untreated plants. Themethod may comprise increasing the biomass of the treated plantscompared to untreated plants. The method may comprise increasing thefruit production of the treated plants compared to untreated plants. Themethod may comprise increasing the production period of the treatedplants compared to untreated plants. The method may comprise increasingthe productive lifespan of the treated plants compared to untreatedplants. The method may comprise using the method with plants grown underhydroponic conditions. The method may comprise using the method withplants grown in a greenhouse. The method may comprise using the methodwith plants grown in a field or outside. The method may comprise usingthe method with plants grown in aeroponic conditions, or combinedhydroponic and aeroponic conditions. The method may comprise using themethod with vertical farming.

A method using a composition of the present invention comprises a methodof treating a surface comprising providing a microorganism-containingcomposition to a surface, wherein the microorganism-containingcomposition comprises at least one isolated bacteria, at least oneisolated yeast, and at least one isolated micorrhyzal fungus, whereinthe bacteria comprise at least a lactobacillus and at least a bacillus,wherein the bacteria were selected based on enzyme profiles so that thebacteria are complementary; and allowing microorganisms of themicroorganism-containing composition to grow and reproduce for a periodof time, wherein the microorganisms compete with endogenous bacteria(whether originally present or added over time from the environment) forat least one environmental factors comprising physical space, nutrients,water, oxygen, or light or chemical energy sources, or combinationsthereof, or wherein the microorganisms of the microorganism-containingcomposition predate and remove microorganisms originally present on thesurface. The method may comprise lowering the number of originallypresent microorganisms. The Lactobacillus may be L. helveticus, or theLactobacillus may be L.helveticus, IN-LH1, Accession No. PTA-12386. TheBacillus may be B. Subtilis, or the Bacillus may be B. subtilis, IN-BS1,Accession No. PTA-12385. The yeast may be Saccharomyces cerevisiae, orthe yeast may be Saccharomyces cerevisiae, referred to as IN-SC1,Accession No. PTA-12384. The microorganism-containing composition mayfurther comprise an isolated photosynthetic bacteria. The photosyntheticbacteria may be Rhodopseudomonas palustris, or the photosyntheticbacteria is Rhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387.The microorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method using a composition of the present invention comprises treatinga fluid, comprising, providing a microorganism-containing composition toa fluid, wherein the microorganism-containing composition comprises atleast one isolated bacteria, at least one isolated yeast, and at leastone isolated micorrhyzal fungus, wherein the bacteria comprise at leasta lactobacillus and at least a bacillus, wherein the bacteria wereselected based on enzyme profiles so that the bacteria arecomplementary; and allowing microorganisms of themicroorganism-containing composition to grow and reproduce for a periodof time, wherein the microorganisms compete with endogenous bacteria(whether originally present or added over time from the environment) forat least one environmental factors comprising physical space, nutrients,water, oxygen, or light or chemical energy sources, or combinationsthereof, or wherein the microorganisms of the microorganism-containingcomposition predate and remove microorganisms originally present in thefluid. The method may comprise lowering the number of coliforms in theliquid. The Lactobacillus may be L. helveticus, or the Lactobacillus maybe L.helveticus, IN-LH1, Accession No. PTA-12386. The Bacillus may be B.subtilis, or the Bacillus may be B. subtilis, IN-BS1, Accession No.PTA-12385. The yeast may be Saccharomyces cerevisiae, or the yeast maybe Saccharomyces cerevisiae, referred to as IN-SC1, Accession No.PTA-12384. The microorganism-containing composition may further comprisean isolated photosynthetic bacteria. The photosynthetic bacteria may beRhodopseudomonas palustris, or the photosynthetic bacteria isRhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387. Themicroorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method using a composition of the present invention comprises makingan article comprising a microorganism-containing composition,comprising, adding a microorganism-containing composition to a surfaceof an article, wherein the microorganism-containing compositioncomprises at least one isolated bacteria, at least one isolated yeast,and at least one isolated micorrhyzal fungus, wherein the bacteriacomprise at least a lactobacillus and at least a bacillus, wherein thebacteria were selected based on enzyme profiles so that the bacteria arecomplementary. The method may comprise steps where themicroorganism-containing composition and surface are dried to attach themicroorganism-containing composition to the surface. The method maycomprise steps where the surface is treated to aid in attachment of themicroorganism-containing composition. The method may comprise adding oneor more components to the microorganism-containing composition to aid inits attachment to the surface. The method may comprise adding one ormore components to both the microorganism-containing composition and thesurface to aid in attachment of the microorganism-containing compositionto the surface. Such components may be any material, compound ormolecule that aids in the attachment of the microorganism-containingcomposition to the surface or the article. For example, glues, starches,natural materials, polymeric materials and materials that are known forattaching microorganisms to surfaces or articles. The method maycomprise surfaces or articles wherein the surface or article is is aglass bead, inert materials, woven materials, nonwoven materials,natural materials such as plant material, coco mats, silica beads,polymeric materials, plant container, container, filter structures,porous inert particles, or zeolites. Articles made with attachedmicroorganism-containing compositions of the present invention arecontemplated by the invention. The Lactobacillus may be L. helveticus,or the Lactobacillus may be L.helveticus, IN-LH1, Accession No.PTA-12386. The Bacillus may be B. subtilis, or the Bacillus may be B.subtilis, IN-BS1, Accession No. PTA-12385. The yeast may beSaccharomyces cerevisiae, or the yeast may be Saccharomyces cerevisiae,referred to as IN-SC1, Accession No. PTA-12384. Themicroorganism-containing composition may further comprise an isolatedphotosynthetic bacteria. The photosynthetic bacteria may beRhodopseudomonas palustris, or the photosynthetic bacteria isRhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387. Themicroorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplanterum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method using a composition of the present invention comprises a methodfor making a food supplement, comprising , growing amicroorganism-containing composition to a particular cellularconcentration, wherein the microorganism-containing compositioncomprises at least one isolated bacteria, at least one isolated yeast,and at least one isolated micorrhyzal fungus, wherein the bacteriacomprise at least a lactobacillus and at least a bacillus, wherein thebacteria were selected based on enzyme profiles so that the bacteria arecomplementary, and isolating factors from the microorganisms or themedia. Factors made by microorganisms that useful as food supplementsare known and methods for isolating them are known to those skilled inthe art. The Lactobacillus may be L. helveticus, or the Lactobacillusmay be L.helveticus, IN-LH1, Accession No. PTA-12386. The Bacillus maybe B. subtilis, or the Bacillus may be B. subtilis, IN-BS1, AccessionNo. PTA-12385. The yeast may be Saccharomyces cerevisiae, or the yeastmay be Saccharomyces cerevisiae, referred to as IN-SC1, Accession No.PTA-12384. The microorganism-containing composition may further comprisean isolated photosynthetic bacteria. The photosynthetic bacteria may beRhodopseudomonas palustris, or the photosynthetic bacteria isRhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387.

The microorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplanterum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method using a composition of the present invention comprises a methodfor making a food supplement, comprising growing amicroorganism-containing composition to a particular cellularconcentration, wherein the microorganism-containing compositioncomprises at least one isolated bacteria, at least one isolated yeast,and at least one isolated micorrhyzal fungus, wherein the bacteriacomprise at least a lactobacillus and at least a bacillus, wherein thebacteria were selected based on enzyme profiles so that the bacteria arecomplementary, and drying and packaging the composition for use as afood supplement. Food supplements comprising microorganisms are knownand methods for preparing and packaging them are known to those skilledin the art. The Lactobacillus may be L. helveticus, or the Lactobacillusmay be L.helveticus, IN-LH1, Accession No. PTA-12386. The Bacillus maybe B. subtilis, or the Bacillus may be B. subtilis, IN-BS1, AccessionNo. PTA-12385. The yeast may be Saccharomyces cerevisiae, or the yeastmay be Saccharomyces cerevisiae, referred to as IN-SC1, Accession No.PTA-12384. The microorganism-containing composition may further comprisean isolated photosynthetic bacteria. The photosynthetic bacteria may beRhodopseudomonas palustris, or the photosynthetic bacteria isRhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387. Themicroorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method using a composition of the present invention comprises a methodfor food supplementation, comprising adding to animal, plant or humanfood, a microorganism-containing composition comprising, isolatedbacteria, at least one isolated yeast, and at least one isolatedmicorrhyzal fungus, wherein the bacteria comprise at least one of alactobacillus, and at least one of a bacillus, wherein the bacteria wereselected based on enzyme profiles so that the bacteria arecomplementary. The Lactobacillus may be L. helveticus, or theLactobacillus may be L.helveticus, IN-LH1, Accession No. PTA-12386. TheBacillus may be B. subtilis, or the Bacillus may be B. subtilis, IN-BS1,Accession No. PTA-12385. The yeast may be Saccharomyces cerevisiae, orthe yeast may be Saccharomyces cerevisiae, referred to as IN-SC1,Accession No. PTA-12384. The microorganism-containing composition mayfurther comprise an isolated photosynthetic bacteria. The photosyntheticbacteria may be Rhodopseudomonas palustris, or the photosyntheticbacteria is Rhodopseudomonas palustris, IN-RP1, Accession No, PTA-12387.The microorganism-containing composition comprises a mixed culture ofisolated microorganisms. A mixed culture may be IN-M1, Accession NoPTA-12383. A mixed culture may comprise Rhodopseudomonas palustris,Bacillus subtilis, Pseudomonas aeruginosa, Saccharomyces cerevisiae,Aspergillus oryzae, Candida utilis, Streptococcus lactis, Lactobacillisplantarum, Lactobacillis helveticus, and Lactobacillus casei, or amicroorganism-containing composition may comprise one or more of themicroorganisms disclosed herein.

A method of the present invention comprises a method for making a mixedculture, comprising, measuring one or more enzyme activities of aplurality of types or species of microorganisms to form an enzymeprofile for each microorganism, wherein an enzyme profile comprises oneor more enzymes and the level of activity for the one or more enzymesfor a particular substrate; selecting two or more microorganisms of a)wherein the enzyme profile of each selected microorganism is notidentical to the enzyme profile of another microorganism so that atleast one enzyme and /or at least one enzyme activity of the enzymes ofthe profile is found in only one microorganism in the mixed culture,growing the selected microorganisms in media to form an incubationmixture. The method may comprise measuring characteristics of theincubation mixture. Measuring characteristics of the incubation mixturemay comprise measuring the predation of one or more selectedmicroorganism by one or more other selected microorganisms, measuringfactors or proteins released or made by one or more microorganisms, pHchanges, or extracellular enzymes excreted by one or more selectedmicroorganisms and its effects on one or more other selectedmicroorganism, measuring the number of cells per mL of one or moreselected microorganism in the mixture, determining growth rate for oneor more selected microorganisms, or combinations of characteristics andmeasurements. The method may comprise using the measured characteristicsof the incubation mixture to determine if one or more microorganisms areto be removed from the incubation mixture or if one or moremicroorganisms are to be added to the incubation mixture. The method maycomprise wherein when one or more microorganisms are to be removed fromthe incubation mixture, the method may comprises killing the incubationmixture and repeating the steps to make a new incubation mixture withoutthe identified unwanted microorganism. The method may comprise stepswherein when one or more microorganisms are to be added to theincubation mixture, the method may comprise adding one or more desiredisolated microorganisms to the incubation mixture, and growing themixture to a predetermined cellular concentration to produce a mixedculture, and optionally, packaging all or a portion of the mixedculture. The method may comprise repeating the steps of the method oneor more times. The method may comprise growing the cells of theincubation mixture to a particular concentration to form an individualmixed culture. The method may comprise making one or more mixed culturesand f) combining the individual mixed cultures to form a mixed culture.The method may comprise growing the mixed culture to a particularconcentration; and packaging all or portions of the mixed culture. Themethod may comprise growing the mixed culture to form an incubationmixed culture and measuring characteristics of the incubation mixedculture. The method may comprise steps of measuring characteristics ofthe incubation mixed culture comprises measuring the predation of one ormore selected microorganism by one or more other selectedmicroorganisms, measuring factors, pH changes, or extracellular enzymesexcreted by one or more selected microorganisms and its effects on oneor more other selected microorganism, measuring the number of cells permL of one or more selected microorganism in the mixture, determinegrowth rate for one or more selected microorganisms, or combinations ofmeasurements. The method may comprise using the measured characteristicsof the incubation mixed culture to determine if one or moremicroorganisms is to be removed from the incubation mixed culture or ifone or more microorganisms is to be added to the incubation mixedculture, or if no change in microorganisms is to be made. The method maycomprise steps for when one or more microorganisms are to be removedfrom the incubation mixed culture, the method further comprises killingthe incubation mixed culture; and repeating the steps to make a newincubation mixture without the undesired one or more microorganisms. Themethod may comprise repeating steps of the method one or more times. Themethod may comprise steps wherein when one or more microorganisms are toadded to the incubation mixed culture, the method further adding one ormore isolated microorganisms to the incubation mixed culture, andgrowing the incubation mixed culture to a predetermined cellularconcentration to produce a mixed culture, and optionally, packaging allor a portion of the mixed culture.

EXAMPLES Example 1 A Method for Making a Microbial Composition

A microorganism, such as a bacteria or yeast, was selected for inclusionin the composition, based on its enzyme activity profile, its ability togrow in media, its lack of spore formation, or other criteria describedherein. The microorganism was grown in standard medium for that organismand when at an exponential growth phase, was aliquoted and stored. Themedia for growing microorganisms, such as yeasts and bacteria, are knownto those skilled in the art.

For example, in making I-M Lab, an aliquot (5 mL of cells at 1×10⁶) ofeach of IN-LH1, IN-BS1 and l.casei were added to a media suitable forlactobacilli and bacillus, such as Water700 ml, Molasses 37,5 g,bentonite clay, 3.75 g, Sea salt 3,75 g, Water 720 ml. The bacteria weregrown to an OD 0.752.

In making I-M PNSB, an aliquot (5 mL of cells at 1×10⁶) of IN-RP1 wasadded to a media suitable for phototrophic bacteria. For example Water134 ml, Fish emulsion 9 ml, IN-SC1 culture (1×10⁶) 1 ml, volume wasadjusted to 144 ml with water and the cells were grown to an OD 0.856.

In making I-M Yeast, an aliquot (5 mL of cells at 1×10⁶) of IN-SC1 andA. oryzae (OD 0.3) South River Miso Company, in Conway, Mass., USA wereadded to a media suitable for yeast, such as Water 390 ml, Molasses 1 g,Fish emulsion 29 g, Kelp 9 g, Wheat germ 1 g.

The volume was adjusted to 432 ml with water. Fish emulsion (acommercially available organic soil amendment, from Nutrivert, Dunham,Quebec non-pasteurized), and the bacteria were grown to an OD 0.574.

To make a compositions such as microorganism-containing compositions,such as IN-M1 deposited with ATCC Accession No. PTA-12383, a mixedculture, the three microbial component mixed cultures were used. I-MLab, I-M PNSB and I-M Yeast were added to a medium comprising water,molasses, mineral powder, sea salt and wheat bran as shown below. Thethree microbial component mixtures were added in the percentages shownin the chart below. The seed culture (an initial mixed culture)comprised IN-RP1, IN-BS1, IN-SC1, Aspergillus oryzae, IN-LH1, andLactobacillus caseii and was made under sterile conditions.

Components of Composition % WATER 88.70 MOLASSES 5.00 I-M LAB 2.00 I-MPNSB 2.00 I-M YEAST 1.00 Bentonite clay (Utah) 0.10 SEA SALT(commercially available) 0.10 WHEAT BRAN 0.10 TOTAL 100

The molasses, sea salt, wheat bran and mineral powder were dissolved insome of the warm water and the temperature was kept at 45-50° C. The I-MLAB, the I-M PNSB and I-M Yeast were added together into a separatecontainer and blended. The total was 50 L, of which 20 L was I-M LAB, 20L was I-M PNSB, and 10 L was I-M Yeast (the composition comprising thesethree microbial compositions may be referred to herein as a seedculture). This seed culture was added to the main tank of media andwater was added to make 110 L, and the temperature was kept at 37° C.with light agitation until the pH is pH 4.0 and below.

A secondary fermentation culture (a mixed culture) was made to produce astable concentrated culture (mixed culture) comprising approx. 1 billionmicroorganisms per liter (1×10⁶ cells/mL). A concentrated compositionmay have a shelf life of 3 years or more. A typical 1000 liter secondaryfermentation batch, was inoculated with 50 litres of the seed culture(described above—20 L was I-M LAB, 20 L was I-M PNSB, and 10 L was I-MYeast) and the media was 50-200 liters of non-sulphur agriculturalmolasses, 3.75 liters wheat bran, (0.02-0.05% by volume), 3.75 literskelp, (0.02-0.05% by volume), 3.75 liters bentonite clay, (0.02-0.05% byvolume), 1.25 liters fish emulsion (a commercially available organicsoil amendment, from Nutrivert, Dunham, Quebec non-pasteurized, 1.25liters soy flour, (0.005-0.03% by volume), 675 mg commercially availablesea salt, and enough non-chlorinated warm water to make 1000 L.

The pH dropped to about 3.7 by Day 5 after inoculation, and the culturewas grown and stirred lightly once per day and pH was monitored. Theculture was incubated for 6 weeks, resulting in themicroorganism-containing composition used in the following examples. Thecomposition was bottled and stored under anoxic conditions in airtightcontainers out of sunlight at room temperature. This resultingcomposition may be referred to as a concentrated composition, with cellsat 1×10⁶ cells/mL.

In an alternative method, the secondary fermentation may contain one ormore strains of microorganisms, such as those purchased from commercialentities, and/or endogenous microorganisms or microbial consortiaisolated from an environment to be treated or a site to be remediated,or a similar environment or site, and may include one or morepollutant(s)or contaminants found in a polluted or contaminated site. Bysimilar site, it is meant that the similar site has the samecontaminants, or contaminants that are chemically or physically similarto ones found in the site to be treated, remediated or to which it isdesired to apply or provide a composition of the present invention.

Example 2 Use of the Compositions and Methods to Control and Reduce PondSludge

On day 1, a 50 liter (5×10⁶ cells per ml) a composition of the presentinvention, which was the result of inducing growth in a closed containerof 80 Liters water plus 10 litres of molasses and an aliquot of 10 Ls ofthe concentrated composition of Example 1, was poured into the pond at adilution of approximately 100:1-1:10000 and was circulated and aeratedvia a pump for three hours per day. On day 14, a second 50 liters of thecomposition above was added to the pond and the body of water wascirculated and aerated as before. On day 25, the duck weed began to dieoff and could be removed from the surface of the pond into the gullywith a hose. Odor from the sediment also disappeared. On day 30, thepond was clear of algae and duck weed. Past day 30, the remediationprocess continued to improve water chemistry, lowering phosphate andnitrogen levels. Furthermore, the water clarity improved, there were noodors present, and the organic sludge continued to float to the surfaceof the pond as it was digested by the microorganisms and could beremoved easily.

Example 3 Use of Microorganism-Containing Composition and Methods toControl Odor Caused by Shallow Pond Sludge

Two shallow interconnecting ponds were inoculated with the concentratedcomposition of Example 1. In the upper pond, concentrated secondaryfermentation product was diluted 1:100 in water and 15 liters weresprayed on the surface and edges of the pond. No aeration was used. Inthe lower pond, 10 liters was sprayed on the pond surface and edges ofthe pond, and, again, no aeration was used. In the adjacent septicseepage field, 5 liters of the concentrated composition of Example 1 wassprayed on the ground and grass in and surrounding the field. Within onehour, odors subsided and continued to be controlled. Further spraying ofthe see page field was done as needed, and, during the following weeks,the water in the ponds clarified.

Example 4 Use of the Compositions and Methods to Control Odor Caused byPond Sludge

A concentrated (1×10⁶ cells/mL) secondary fermentation composition ofExample 1, was diluted 1:100 in water and 30 liters were sprayed on thesurface and edges of the pond. No aeration was used. On day 14, 30liters of pond water from the pond to be treated was blended with 30 mlconcentrated secondary fermentation composition of Example 1 incubatedat ambient temperature for 5 days and then sprayed on the pond surfaceand edges of the pond. No aeration was used. Within 3 hours of theinitial inoculation odors subsided and continued to be controlled. Thecontrol of odor and improvement in water clarity continued throughoutthe summer and autumn following the treatment.

Example 5 Use of the Compositions and Methods to Remediate Soil

A variation of a concentrated (1×10⁶ cells/mL) composition of Example 1,where no pseudomonas was included in the original composition, wasdiluted 1:100 in water, and 5 gallons per acre was sprayed onto the teeboxes, greens, and fairways of a golf course. Sensitive areas such astees and greens, as well as hot spots or brown spots, were sprayed onceor twice per week with the concentrated secondary fermentation productor a variation of the microorganism-containing composition that wasdiluted 1:100 in water. Seven days after the application of the product,brown spots on putting greens were no longer visible.

Example 6 Use of the Compositions and Methods in the Raising of Animals

Dogs were given the concentrated (1×10⁶ cells/mL) composition of Example1 as an additive in their food, twice daily, at a dose of 1% by weightof the food. The administration of the concentrated composition ofExample 1 led to improvement in body odor, reduced flatulence, whiteningof the teeth, and reduced symptoms of gingivitis, including a reductionof tarter as well as redness of the gums. The dogs were also given theconcentrated composition of Example 1 externally as a shower/rinse onceper week at a dose of 3% by volume. The use of the concentratedcomposition of Example 1 led to improved skin condition and the controlof bad odors from the animal's coat.

Cats were given the concentrated (1×10⁶ cells/mL) composition of Example1 as an additive in their food, twice daily, at a dose of 1% of the foodby weight. The administration of the concentrated composition of Example1 improved bad breath, and resulted in a softer and shinier coat.

Indoor farm animal enclosures such as barns, stalls, litter woodshavings, and wood pellets were sprayed with the concentratedcomposition of Example 1, at a concentration of 3% by volume. During thefirst week of treatment, the enclosures were sprayed with theconcentrated composition of Example 1 twice daily. During the secondweek of treatment, the enclosures were sprayed with the product oncedaily. Thereafter, the enclosures were sprayed with the concentratedcomposition of Example 1 on a weekly basis. Use of the concentratedcomposition of Example 1 resulted in a reduction of odors in theenclosures and facilitated in the cleaning of stalls and otherenvironments.

Horses were given 50 cc of the concentrated (1×10⁶ cells/mL) compositionof Example 1 daily, at a dose of 3% dilution by weight in beet pulp. Useof the concentrated composition of Example 1 resulted in improved coats,which became shinier within 3 to 7 days. Furthermore, urine becameclearer within 2 weeks of use, and manure degraded faster. Additionally,the horses were given the composition of Example 1 externally as ashower/rinse at a dose of 3% by volume. External use kept black houseflies away from the horses, and horses that were nervous of beingsprayed with standard or natural insecticides showed no reaction to theuse of the product.

A parrot was given the concentrated (1×10⁶ cells/mL) composition ofExample 1 externally as a spray at a dose of 1% dilution by volume. Noadverse effects were observed from preening after administration of theconcentrated composition of Example 1. The concentrated composition ofExample 1 was also used to spray the parrot's cage. Use of thecomposition of Example 1 resulted in a reduction of odors and dust fromthe cage.

Example 7 Use of the Compositions and Methods to Treat and Maintain aResidential Septic System

A family with a residential septic system, having a flow rate of 1200liters per day, began to experience problems with odors backing up intothe residence and outside of the residence as the effluent was pumpedinto the seepage field. Upon inspection, it was noted that a 30 cm crustof bio-solids was floating on the top of the drop out tank, and therewere blockages in the in-flow and out-flow pipes. 250 ml of theconcentrated (1×10⁶ cells/mL) composition of Example 1 was added afterbreaking through the floating crust. Additionally, a simple aerationdevice was put into the drop out tank. An immediate reduction of odorwas noticed in the residence. After 16 hours, there were no odorsemanating from the pump or the seepage field. Moreover, the bio-solidcrust was digested. To conclude treatment of the system, a furtherinoculation of 500 ml of the concentrated composition of Example 1 wasconducted after 8 weeks.

Alternative methods for the treatment of septic sludge may include afeed and grow activation prior to treatment; this may include additionalmolasses or another carbon source.

Example 8 Soil Testing on Golf Turf Increased Phosphate Content

On a golf green, half of the green was treated one time per week withIN-M1, Accession No. PTA-12383 (3.8×10⁹cells per L in chlorine freewater for 5 weeks and the green was included in the standard turfmanagement program of the course. The green was treated as normallytreated with chemical fertilizers and pesticides. A soil analysis wasperformed for the treated half (treated with Inocul-M) and the untreatedhalf

Phosphate Untreated 82 ppm Treated 117 ppm   30% Potassium Untreated 65ppm Treated 70 ppm 7.11% Magnesium Untreated 78 ppm Treated 86 ppm  9.3%

Measurements taken after treatment with a microorganism-containingcomposition, IN-M1, Accession No. PTA-12383 showed an increase inphosphate in the soil.

Example 9 Treatment of Wastes and Removal Of Coliforms

This study was designed to monitor effects on coliform contamination ofseptic sludge from city municipality waste water treatment plant withIN-M1 at 1×10⁶ cells/mL and to monitor a wide range of endpointsincluding coliform, toxic chemical and organic compounds. It was foundthat there was a significant reduction in coliform presence (measured ascfu/100mL, colony forming units), from 8.3 million cfus to 100,000cfus/100 ml, following 7 days of treatment. The entire treatment periodwas 22 days. There was an increase in nitrate concentration whichindicates an increase in nutritional value of the biosolids as a soilamendment for agricultural purposes. There was a trend in reduction ofmercury concentration and change in toluene concentration.

Protocol Per Cubic Meter Septic Sludge

0.1 L concentrate for example, IN-M1 at 1×10⁶ cells/mL, +0.2 L molassas+0.75 L water per m³ sludge were combined together. The mixture wasallowed to stand at ambient temperature for 48 hours. The mixture wasthoroughly combined with a 2-4% septic sludge and water in the lagoon orsettling tank of approximately 2000 liters. In the case of separatedhigh percentage septic sludge, the sample should be mixed verythoroughly making sure that the product is distributed throughout toavoid pockets where pathogens can grow. The lagoon was left alone for2-6 weeks.

Though not wishing to be bound by any particular theory, in a watermixture, the bio-solids gradually diminished in quantity, and it isthought that the beneficial microbes colonized the water and sedimentand dissolved the organic matter; at the same time the pre-biotics andsignaling peptides stimulated the indigenous flora to also breakdown theorganic material. In another instance coli-form bacteria replication wasinhibited by the addition of 0.1 L of a concentrated solution ofIN-M1,+0.2 L molasses+0.75 L water per m³ sludge were combined togetherand left to ferment at ambient temperature 22 days. The composition maycontain species which sequester nitrogen both from volatile organiccompounds (VOCs) and the air and convert it into soluble forms. See FIG.4.

Detection Sample Day 0 Day 7 Day 22 limit Coliforms 8.2 × 10⁶ 4.3 × 10⁵ 1.0 × 10⁵ 1 (cfu/100 mL) Ammonia mg/L 870 360 421 5 Nitrate 0.5 32.11.32 × 10⁴ 0.5 Mercury 0.351 0.278 0.255 0.05 Toluene mg/L 0.432 0.2440.373 0.005

The decrease in ammonia bound nitrogen decreased by 58.6% day 7 to 51.6%day 22; nitrate bound levels increased more than 6000 times by treatmentday 7 to approximately 2.6 million times by day 22. This may be evidenceof either transfer of nitrogen from volatile form (ammonia) to solubleform (nitrates) and/or the sequestering of nitrogen to a soluble formfrom the air, through the action of nitrogen fixing microbes in theformulation. The treatment may enhance the rapid conversion of nitrogento soluble forms which may be used as a soil amendment. It is thoughtthat the relative nitrogen mass difference is due to the fact thefermentation parameters are anoxic. See FIGS. 3A and B. See FIGS. 4 and5 for mercury and toluene results.

1-117. (canceled)
 118. A composition comprising one or moremicroorganisms, wherein the microorganism is IN-M1, Accession No.PTA-12383.
 119. The composition of claim 118, further comprisingfermentation broth of the microorganism.
 120. An article comprising thecomposition of claim
 118. 121. The article of claim 120, wherein thearticle is a glass bead, inert material, woven material, nonwovenmaterial, plant material, coco mat, silica bead, polymeric material,plant container, container, filter structure, porous inert particle, orzeolite.
 122. A method to improve soil quality, the method comprisingthe step of applying an effective amount of the composition of claim 118to soil; wherein the quality of the soil is improved compared tountreated soil.
 123. The method of claim 122, wherein the soil qualityimproved is water retention.
 124. The method of claim 122, wherein thesoil quality improved is soil aeration.
 125. A method to improve soilquality, the method comprising the step of applying an effective amountof the article of claim 121 onto soil; wherein the quality of the soilis improved compared to untreated soil.
 126. The method of claim 125,wherein the soil quality improved is water retention.
 127. The method ofclaim 125, wherein the soil quality improved is soil aeration.
 127. Acomposition comprising one or more microorganism, wherein themicroorganism is IN-M1, Accession No. PTA-12383; and fermentation brothof the microorganism.
 128. An article comprising the composition ofclaim
 127. 129. The article of claim 128, wherein the article is a glassbead, inert material, woven material, nonwoven material, plant material,coco mat, silica bead, polymeric material, plant container, container,filter structure, porous inert particle, or zeolite.
 130. A method toimprove soil quality, the method comprising the step of applying aneffective amount of the composition of claim 127 onto soil; wherein thequality of the soil is improved compared to untreated soil.
 131. Themethod of claim 130, wherein the soil quality improved is waterretention.
 132. The method of claim 130, wherein the soil qualityimproved is soil aeration.
 133. A method to improve soil quality, themethod comprising the step of applying an effective amount of thearticle of claim 128 onto soil; wherein the quality of the soil isimproved compared to untreated soil.
 134. The method of claim 134,wherein the soil quality improved is water retention.
 135. The method ofclaim 135, wherein the soil quality improved is soil aeration.